U.S. patent application number 13/948271 was filed with the patent office on 2014-01-23 for endpoint zygosity assay to detect rf4 gene in maize.
The applicant listed for this patent is Dow AgroSciences LLC. Invention is credited to Siva Prasad KUMPATLA, Ruihua REN, Peizhong ZHENG.
Application Number | 20140024037 13/948271 |
Document ID | / |
Family ID | 48917713 |
Filed Date | 2014-01-23 |
United States Patent
Application |
20140024037 |
Kind Code |
A1 |
REN; Ruihua ; et
al. |
January 23, 2014 |
Endpoint Zygosity Assay To Detect RF4 Gene In Maize
Abstract
A method is provided for determining the zygosity of an Rf4 gene
in a corn plant. A method may include performing a first PCR assay,
a second PCR assay, quantifying probes used in the first and second
PCR assays, and comparing the quantified probes to determine
zygosity.
Inventors: |
REN; Ruihua; (Carmel,
IN) ; ZHENG; Peizhong; (Carmel, IN) ;
KUMPATLA; Siva Prasad; (Carmel, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dow AgroSciences LLC |
Indianapolis |
IN |
US |
|
|
Family ID: |
48917713 |
Appl. No.: |
13/948271 |
Filed: |
July 23, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61674556 |
Jul 23, 2012 |
|
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|
Current U.S.
Class: |
435/6.12 ;
536/24.33 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C12Q 1/6895 20130101; C12Q 2600/13 20130101 |
Class at
Publication: |
435/6.12 ;
536/24.33 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A PCR assay method for determining zygosity of an Rf4 gene in a
corn plant, the method comprising: performing a first PCR assay
using a first probe, a first forward primer, and a first reverse
primer on a polynucleotide from a corn plant sample; performing a
second PCR assay using a second probe, a second forward primer, and
a second reverse primer on the polynucleotide sample; quantifying
the first and second probe; and, comparing the quantified first and
second probe to determine zygosity.
2. The PCR assay method of claim 1, wherein the PCR assay is a
multiplex PCR-format.
3. The PCR assay method of claim 2, wherein the first and second
PCR assays are performed in a single PCR assay tube.
4. The PCR assay method of claim 1, wherein the PCR is real-time
PCR.
5. The PCR assay method of claim 1, further comprising: loading a
PCR reaction mixture in a PCR assay tube; wherein the PCR reaction
mixture comprises a polymerase with 5' to 3' nuclease activity,
deoxynucleotides, a buffer, the first and second forward primer,
the first and second reverse primer, the first and second probe,
and the polynucleotide sample, and the first probe and the second
probe comprising fluorescent dyes with distinguishable
excitation/emission spectra; and performing an amplification step
under amplification conditions such that the 5' to 3' nuclease
activity of the polymerase cleaves the first and second probe of
claim 1, thereby releasing fluorescent dyes comprising
distinguishable excitation/emission spectra.
6. The PCR assay method of claim 1, further comprising: loading a
PCR reaction mixture in a PCR assay tube, wherein the PCR reaction
mixture comprises a polymerase with 5' to 3' nuclease activity,
deoxynucleotides, a buffer, the first or second forward primer, the
first or second reverse primer, the first or second probe, and the
polynucleotide sample; and performing an amplification step under
conditions such that the 5' to 3' nuclease activity of the
polymerase cleaves the first or second probe, thereby releasing
fluorescent dyes comprising distinguishable excitation/emission
spectra.
7. The PCR assay method of claim 1, wherein the first and second
probes are quantified by measuring excitation/emission spectra
emitted from the fluorescent dyes, during the amplification.
8. The PCR assay method of claim 1, wherein zygosity is determined
by comparing the quantified first and second probe using a
.DELTA..DELTA.Ct formula.
9. The PCR assay method of claim 1, wherein the first forward
primer comprises SEQ ID NO:1.
10. The PCR assay method of claim 1, wherein the first reverse
primer comprises SEQ ID NO:2.
11. The PCR assay method of claim 1, wherein the probe comprises
SEQ ID NO:3.
12. The PCR assay method of claim 1, wherein the probe comprises a
first fluorescent dye and a first quencher.
13. The PCR assay method of claim 12, wherein the first fluorescent
dye comprises a HEX fluorescent dye, a VIC fluorescent dye, a FAM
fluorescent dye, a JOE fluorescent dye, a TET fluorescent dye, a Cy
3 fluorescent dye, a Cy 3.5 fluorescent dye, a Cy 5 fluorescent
dye, a Cy 5.5 fluorescent dye, a Cy 7 fluorescent dye, or a ROX
fluorescent dye.
14. The PCR assay method of claim 12, wherein the quencher
comprises a Dabcyl quencher, a Tamra quencher, a Qx1 quencher, an
Iowa Black FQ quencher, an Iowa Black RQ quencher, an IR Dye QC-1
quencher, a MGB quencher, or a Blackhole quencher.
15. The PCR assay method of claim 12, wherein the first probe
comprises FAM as the first fluorescent dye at the 5' end of the
first probe and MGB as the first quencher on the 3' end of the
first probe.
16. The PCR assay method of claim 1, wherein the second forward
primer comprises SEQ ID NO:1.
17. The PCR assay method of claim 1, wherein the second reverse
primer comprises SEQ ID NO:2.
18. The PCR assay method of claim 1, wherein the second probe
comprises SEQ ID NO:4.
19. The PCR assay method of claim 18, wherein the second probe
comprises a second fluorescent dye and a second quencher.
20. The PCR assay method of claim 19, wherein the second
fluorescent dye comprises a HEX fluorescent dye, a FAM fluorescent
dye, a VIC fluorescent dye, a JOE fluorescent dye, a TET
fluorescent dye, a Cy 3 fluorescent dye, a Cy 3.5 fluorescent dye,
a Cy 5 fluorescent dye, a Cy 5.5 fluorescent dye, a Cy 7
fluorescent dye, or a ROX fluorescent dye.
21. The PCR assay method of claim 19, wherein the second quencher
comprises a Dabcyl quencher, a Tamra quencher, a Qx1 quencher, an
Iowa Black FQ quencher, an Iowa Black RQ quencher, an IR Dye QC-1
quencher, a MGB quencher, or a Blackhole quencher.
22. The PCR assay method of claim 19, wherein the second probe
comprises VIC as the second fluorescent dye at the 5' end of the
second probe, and MGB as the second quencher on the 3' end of the
second probe.
23. The PCR assay method of claim 1, wherein the Rf4 gene comprises
a dinucleotide substitution at nucleotide positions 1664-1665 as
Rf4-bHLH genomic sequences indicated above (at amino acid positions
186-187).
24. The PCR assay method of claim 1, wherein the corn plant
comprises an Rf4 dinucleotide substitution at nucleotide positions
1664-1665 as Rf4-bHLH genomic sequences indicated above (at amino
acid positions 186-187).
25. The PCR assay method of claim 1, wherein the corn plant is a
maize germplasm.
26. The PCR assay method of claim 1, wherein the corn plant
comprises a plant part, plant organ, plant seed, or plant cell.
27. The PCR assay method of claim 26, wherein the plant part is
selected from the group consisting of leaves, pollen, embryos,
cotyledons, hypocotyls, meristematic cells, roots, root tips,
anthers, flowers, stems, and pods.
28. The PCR assay method of claim 1, wherein the method is used to
determine the presence or absence of the Rf4 allele in a
polynucleotide sample.
29. The PCR assay method of claim 1, wherein the method is used for
breeding introgression into a second line of Zea mays.
30. The PCR assay method of claim 29, wherein the second line of
Zea mays does not contain the Rf4 allele.
31. The PCR assay method of claim 29, wherein the method is used to
detect the presence or absence of the Rf4 allele within progeny
plants.
32. A DNA detection kit for performing the breeding introgression
method of claim 29, the kit comprising a forward primer (SEQ ID
NO:1), a reverse primer (SEQ ID NO:2), a first probe (SEQ ID NO:3),
and a second probe (SEQ ID NO:4).
33. The PCR assay method of claim 1, wherein the method is used to
identify lines of Zea mays that possess restored male
fertility.
34. A DNA detection kit for performing the method of claim 1.
35. The DNA detection kit of claim 34, comprising the forward and
reverse primers, SEQ ID NO:1 and SEQ ID NO:2.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e) of U.S. Provisional Application Ser. No. 61/674,556,
filed on Jul. 23, 2012, the entire disclosure of which is
incorporated herein by reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jul. 22, 2013, is named 6257-225987_SL.txt and is 28,606 bytes
in size.
BACKGROUND
[0003] Cytoplasmic male sterility (CMS) is a maternally inherited
inability to produce functional pollen and has been successfully
used in commercial production of hybrid seed, avoiding the
drawbacks of hand or mechanical emasculation (Kaul, 1988). Breeders
produce hybrid seed using a CMS system by developing female lines
that carry CMS cytoplasm but lack restorer genes and by developing
male lines that carry the appropriate restorer genes. F1 hybrid
seed produced by the female lines carry the CMS cytoplasm but yield
fertile plants because of the action of the paternally contributed
nuclear restorer genes.
[0004] More than 40 sources of CMS have been found in maize and
were classified into three major groups by differential fertility
restoration reactions. These groups are designated as CMS-T
(Texas), CMS-S (USDA) and CMS-C (Charrua) (Beckett 1971). In the
CMS-T group, two dominant genes, Rf1 and Rf2, located on
chromosomes 3 and 9, respectively, are required for the restoration
of pollen fertility (Duvick 1965). The S-cytoplasm is restored by a
single gene, Rf3 on chromosome 2 (Laughnan and Gabay 1978).
[0005] Rf genes have been cloned or mapped to high resolutions from
several plant species, for example, Rf2 from maize (Zea mays) (Cui
et al., 1996), Rf-PPR592 from Petunia (Petunia hybrida) (Bentolila
et al., 2002), Rfo from radish (Raphanus sativus) (Brown et al.,
2003; Desloire et al., 2003; Koizuka et al., 2003), Rf1 and Rf2
from sorghum (Sorghum bicolor) (Klein et al., 2005), Rf1a and Rf1b
from rice (Oryza sativa) for BT-type CMS (Kazama and Toriyama,
2003; Akagi et al., 2004; Komori et al., 2004; Wang et al., 2006),
Rf17 (RMS) from rice (Oryza sativa) for CW-type CMS (Fujii &
Toriyama, 2009), Rf1& Rf2 from monkey flower (Mimulus guttatus)
(Barr & Fishman, 2010). Rf4 for CMS C-type of maize was
recently cloned.
SUMMARY
[0006] Disclosed herein includes methods and reagents for detecting
and quantifying the zygosity of the Rf4 gene in plants. The methods
can employ and the reagents can include primers and oligonucleotide
probes configured for a multiplex, real-time quantitative PCR
(qPCR) assay.
[0007] In an embodiment, the present method can detect and quantify
the zygosity of the Rf4 gene in corn plants in a single reaction.
The method can employ primers and oligonucleotide probes that are
specific and can distinguish between Rf4 alleles.
[0008] In an embodiment, a method for determining zygosity of an
Rf4 gene in a corn plant includes: a) performing a first PCR assay
using a first probe, a first forward primer, and a first reverse
primer on a polynucleotide sample from a corn plant; b) performing
a second PCR assay using a second probe, a second forward primer,
and a second reverse primer on the polynucleotide sample from a
corn plant; c) quantifying the first and second probes; and d)
comparing the quantified first and second probes to determine
zygosity. In an embodiment, the probes are detectably labeled. In
an embodiment, the primers and probes are specific for the Rf4 gene
in a corn plant. In an embodiment, a forward primer specific for
the Rf4 gene comprises SEQ ID NO:1, SEQ ID NO:11, or SEQ ID NO:15.
In an embodiment, a reverse primer specific for the Rf4 gene
comprises SEQ ID NO:2, SEQ ID NO:14, or SEQ ID NO:18. In an
embodiment, a probe specific for the Rf4 gene comprises SEQ ID
NO:4, SEQ ID NO:13, or SEQ ID NO:17.
BRIEF DESCRIPTION OF THE FIGURES
[0009] FIG. 1A. Real-time PCR amplification plots with relative
fluorescence unit (RFU) are shown for Rf4 with FAM and CMS-C.
Exponential amplification phase was observed from cycles 23 to 35
for both Rf4 and CMS-C or non-restorer genes.
[0010] FIG. 1B. Real-time PCR amplification plots with relative
fluorescence unit (RFU) are shown for Rf4 with CMS-C and
non-restorer with VIC. Exponential amplification phase was observed
from cycles 23 to 35 for both Rf4 and CMS-C or non-restorer
genes.
[0011] FIG. 2. Rf4 zygosity test with end-point TaqMan assay using
KLIMS. The raw fluorescence intensity data directly from the plate
reader was analyzed in KLIMS. A graph with RFU of FAM as x-axis and
VIC as y-axis was generated. Zygosity calls were made based on the
cluster separation in a cluster view.
DETAILED DESCRIPTION
Definitions
[0012] The term "sample" refers to a part from any plant species,
but preferably is from maize (Zea mays). Such can be at the macro
or micro level, wherein polynucleotides and/or polypeptides can be
extracted.
[0013] The term "plant" includes reference to whole plants, plant
parts, seeds, plant cells, and progeny of same. Plant parts can
include, but are not limited to, leaves, pollen, embryos,
cotyledons, hypocotyls, meristematic cells, roots, root tips,
anthers, flowers, stems, and pods.
[0014] The term "corn" refers to Zea mays or maize and includes all
plant varieties that can be bred with corn, including wild maize
species.
[0015] As used herein, "buffer" refers to a buffered solution that
resists changes in pH by the action of its acid-base conjugate
components. Buffers may optionally comprise a salt such as
MgCl.sub.2, MnCl.sub.2, or the like. Buffers may also optionally
comprise other constituents to improve the efficiency of reverse
transcription or amplification.
[0016] The term "breeding introgression" refers to the movement of
a gene or genes through sexual crossing, usually by pollen, from a
plant which is intended to be the donor for the formation of
seed.
[0017] The term "allele" refers to an alternative form of a gene,
whereby two genes can differ in DNA sequences. Such differences may
result from at least one mutation (e.g., deletion, insertion,
and/or substitution) in the nucleic acid sequence. Alleles may
result in modified mRNAs or polypeptides whose structure or
function may or may not be modified. Any given gene may have none,
one, or many allelic forms. Each of these types of changes may
occur alone, or in combination with the others, one or more times
in a given sequence.
[0018] The term "zygosity" refers to the similarity of alleles for
a gene or trait in an organism (e.g., a plant). If both alleles are
the same, the organism is homozygous for the allele. If the two
alleles are different, the organism is heterozygous for the gene or
trait. If one allele is not present, the organism is hemizygous. If
both alleles are not present, the organism is nullizygous.
[0019] The term "label" when used herein refers to a detectable
compound or composition that is conjugated directly or indirectly
to a probe to generate a "labeled" probe. The label may be
detectable by itself (e.g. radioisotope labels or fluorescent
labels) or, in the case of an enzymatic label, may catalyze
chemical alteration of a substrate compound or composition that is
detectable (e.g., avidin-biotin).
[0020] The term "oligonucleotide" refers to a single-stranded
nucleic acid including at least between two and about 100 natural
or modified nucleotides or a mixture thereof. The oligonucleotide
can be derived from a natural nucleic acid or produced by chemical
or enzymatic synthesis.
[0021] "Polynucleotide," or "nucleic acid," as used interchangeably
herein, refer to polymers of nucleotides of any length, and include
DNA and RNA. The nucleotides can be deoxyribonucleotides,
ribonucleotides, modified nucleotides or bases, and/or their
analogs, or any substrate that can be incorporated into a polymer
by DNA or RNA polymerase, or by a synthetic reaction. A
polynucleotide may comprise modified nucleotides, such as
methylated nucleotides and their analogs. If present, modification
to the nucleotide structure may be imparted before or after
assembly of the polymer. The sequence of nucleotides may be
interrupted by non-nucleotide components. A polynucleotide may be
further modified after synthesis, such as by conjugation with a
label.
[0022] "Polypeptide" refers to a peptide or protein containing two
or more amino acids linked by peptide bonds, and includes peptides,
oligimers, proteins, and the like. Polypeptides can contain
natural, modified, or synthetic amino acids. Polypeptides can also
be modified naturally, such as by post-translational processing, or
chemically, such as amidation, acylation, cross-linking, and the
like.
[0023] "Polymerase chain reaction" or "PCR" refers to a procedure
or technique in which minute amounts of nucleic acid, RNA and/or
DNA, are amplified as described in U.S. Pat. No. 4,683,195 issued
Jul. 28, 1987. Generally, sequence information from the ends of the
region of interest or beyond needs to be available, such that
oligonucleotide primers can be designed; these primers will be
identical or similar in sequence to opposite strands of the
template to be amplified. The 5' terminal nucleotides of the two
primers may coincide with the ends of the amplified material. PCR
can be used to amplify specific RNA sequences, specific DNA
sequences from total genomic DNA, and cDNA transcribed from total
cellular RNA, bacteriophage or plasmid sequences, etc. See
generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol.,
51:263 (1987); Erlich, ed., PCR Technology, (Stockton Press, NY,
1989).
[0024] The term "primer" refers to an oligonucleotide capable of
acting as a point of initiation of synthesis along a complementary
strand when conditions are suitable for synthesis of a primer
extension product. The synthesizing conditions include the presence
of four different deoxyribonucleotide triphosphates and at least
one polymerization-inducing agent such as reverse transcriptase or
DNA polymerase. These are present in a suitable buffer, which may
include constituents which are co-factors or which affect
conditions such as pH and the like at various suitable
temperatures. A primer is preferably a single strand sequence, such
that amplification efficiency is optimized, but double stranded
sequences can be utilized.
[0025] The term "probe" refers to an oligonucleotide that
hybridizes to a target sequence. In the TaqMan.RTM. or
TaqMan.RTM.-style assay procedure, the probe hybridizes to a
portion of the target situated between the annealing site of the
two primers. A probe can further include a detectable label, e.g.,
a fluorophore (Texas-Red.RTM., Fluorescein isothiocyanate, etc.,).
The detectable label can be covalently attached directly to the
probe oligonucleotide, e.g., located at the probe's 5' end or at
the probes 3' end. A probe including a fluorophore may also further
include a quencher, e.g., Black Hole Quencher.TM., Iowa Black.TM.,
etc. A probe includes about eight nucleotides, about ten
nucleotides, about fifteen nucleotides, about twenty nucleotides,
about thirty nucleotides, about forty nucleotides, or about fifty
nucleotides. In some embodiments, a probe includes from about eight
nucleotides to about fifteen nucleotides.
[0026] The terms "specifically hybridizable" and "specifically
complementary" are terms that indicate a sufficient degree of
complementarity such that stable and specific binding occurs
between the nucleic acid molecule and the DNA target. A nucleic
acid molecule need not be 100% complementary to its target sequence
to be specifically hybridizable. A nucleic acid molecule is
specifically hybridizable when there is a sufficient degree of
complementarity to avoid non-specific binding of the nucleic acid
to non-target sequences under conditions where specific binding is
desired, for example, under stringent hybridization conditions.
Hybridization conditions resulting in particular degrees of
stringency will vary depending upon the nature of the hybridization
method of choice and the composition and length of the hybridizing
nucleic acid sequences. Generally, the temperature of hybridization
and the ionic strength (especially the Na.sup.+ and/or Mg
concentration) of the hybridization buffer will determine the
stringency of hybridization, though wash times also influence
stringency. Calculations regarding hybridization conditions
required for attaining particular degrees of stringency are known
to those of ordinary skill in the art, and are discussed, for
example, in Sambrook et al. (ed.) Molecular Cloning: A Laboratory
Manual, 2.sup.nd ed., vol. 1-3, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1989, chapters 9 and 11; and Hames
and Higgins (eds.) Nucleic Acid Hybridization, IRL Press, Oxford,
1985. Further detailed instruction and guidance with regard to the
hybridization of nucleic acids may be found, for example, in
Tijssen, "Overview of principles of hybridization and the strategy
of nucleic acid probe assays," in Laboratory Techniques in
Biochemistry and Molecular Biolofiy-Hybridization with Nucleic Acid
Probes, Part I, Chapter 2, Elsevier, NY, 1993; and Ausubel et ah,
Eds., Current Protocols in Molecular Biology, Chapter 2, Greene
Publishing and Wiley-Interscience, NY, 1995.
[0027] The term "quenching" refers to a decrease in fluorescence of
a fluorescent detectable label caused by energy transfer associated
with a quencher moiety, regardless of the mechanism.
[0028] The term "reaction mixture" or "PCR reaction mixture" or
"master mix" or "master mixture" refers to an aqueous solution of
constituents in a PCR or RT-PCR reaction that can be constant
across different reactions. An exemplary RT-PCR reaction mixture
includes buffer, a mixture of deoxyribonucleoside triphosphates,
reverse transcriptase, primers, probes, and DNA polymerase.
Generally, template RNA or DNA is the variable in a PCR or RT-PCR
reaction.
Rf4 Gene
[0029] The use of the fertility restorer gene (Rf) with the
cytoplasmic male sterility has been shown to simplify seed
production programs and reduce the overall costs by totally
eliminating manual and machine detasseling. The restoration gene
Rf4 for CMS-C type cytoplasm was previously mapped. The candidate
gene Rf4-bHLH was identified, which encoded a
basic-Helix-Loop-Helix (bHLH) transcription factor. A 3.2-kb genome
DNA fragment was cloned that contained the entire coding region of
the Rf4-bHLH gene plus a 1.1 kb 5' UTR/promoter and a 0.75 kb 3'
UTR/terminator from a CMS-C line (non-restorer), inbred line
(non-CMS) and three restorer lines. Based on nucleotide changes
within Rf4 gene between restorer and non-restorer lines, an
endpoint TaqMan.RTM. PCR assay was developed for Rf4 zygosity test.
The assay was validated with an F2 mapping population which
contained 500 individuals. This assay enables a large scale
screening of maize germplasms in breeding programs for Rf4
restoration gene in a high throughput format. The development of
this assay makes it much easier and cheaper to use the CMS-C/Rf4
system for hybrid corn seed production.
Quantitative PCR
[0030] Quantitative PCR (qPCR) allows automated quantification of
reaction product for each sample per cycle. Commonly used
instrumentation and software products perform the quantification
calculations automatically. The quantification has a broad
10.sup.7-fold dynamic range that is possible, but usually, the
dynamic range is closer to 2-3 logs. Current instrumentation
technology, e.g., Cepheid's Smart Cycler.RTM., allows the
simultaneous detection and quantification of fluorescent signals in
up to four different channels in real-time. In addition, the latest
generation of thermal cyclers is designed to maximize dye
excitation providing a more accurate means of detecting
fluorescence. Thus, multiple amplification products can be assessed
in the same reaction mixture and quantified more accurately
("multiplex PCR" which refers to simultaneous amplification of many
targets of interest in one reaction by using more than one pair of
primers). Further, each reaction site can be programmed
independently, thereby starting the reaction independent of other
reactions. Thus, samples can be evaluated as needed and do not have
to wait for the completion of a programmed reaction already in
progress. Therefore, this new technology now allows for the
detection and quantification of multiple targets in a single sample
in real-time
[0031] There are different probe systems for qPCR (e.g., Molecular
Beacons (Sigma-Genosys, Inc., The Woodlands, Tex.), Scorpions.RTM.
(DxS Ltd., Manchester, UK), SYBR.RTM. Green (Molecular Probes,
Eugene, Oreg.), and TaqMan.RTM. (Applied Biosystems, Foster City,
Calif.). These systems employ fluorescent labels where the
instrumentation detects the fluorescence and the software
interprets levels of fluorescence.
[0032] TaqMan.RTM. utilizes Forster Resonance Energy Transfer
(FRET) by coupling a fluorescent label with a quencher moiety. A
fluorescent label is covalently bound to the 5' end of an
oligonucleotide probe, while the 3' end has a quencher moiety
attached. These oligonucleotide probes are site specific to
hybridize to the amplified product. Preferably, the oligonucleotide
probes are designed to hybridize to a central region of the
amplified product. For TaqMan.RTM. assays, the 5'-nuclease activity
of the DNA polymerase cleaves the probe during the replication
cycle. Due to the cleavage of the probe, the quencher moiety is no
longer coupled to the fluorescence label and cannot quench
fluorescence. Fluorescence thus represents replicating DNA.
Quantification of PCR Results
[0033] Standard Curve.
[0034] Nucleic acids can be used to establish a standard curve.
These methods are well known and include internal controls, double
stranded DNA, a cDNA expressing a target gene, or an in vitro
generated single stranded DNA. Methods may vary according to the
nucleic acid chosen to serve as the standard to establish a
standard curve.
[0035] Comparative Cycle Threshold.
[0036] The comparative cycle threshold (Ct) method, also known as
the 2.sup.-.DELTA..DELTA.Ct method, is also used to quantify DNA
levels. The Ct method compares a test reaction with a control or
calibrator sample. The Ct values of both the control/calibrator
sample and the test sample are normalized. In an embodiment of the
invention, the Ct values were normalized to an arbitrary cutoff,
20-22. In another embodiment, the Ct values were normalized to
within 1 Ct value of a negative control (a sample with no
inhibition). This allows for the sensitivity of the assay and
proper dynamic range.
[0037] The Ct method can also be described by the .DELTA..DELTA.Ct
formula; .DELTA..DELTA.Ct=.DELTA.Ct.sub.test
sample-.DELTA.Ct.sub.reference sample.The amplification
efficiencies of the test sample and the reference sample must be
about the same for the formula to operate. Amplification
efficiencies can be determined by a comparison of the samples with
template dilution. The amplification efficiency is about the same
when a plot of cDNA dilution versus .DELTA.Ct approximates
zero.
End-Point Zygosity Assay
[0038] An end-point PCR assay for testing Rf4 zygosity in a high
throughput way has been developed. This assay enables large scale
and high throughput screening of maize germplasms with the Rf4
restoration gene. This assay will also increase the scale of using
a CMS-C/Rf4 system for hybrid corn seed production.
[0039] In an embodiment, a method for determining the zygosity of
the Rf4 gene in a corn plant includes a PCR assay. Such a PCR assay
can be quantitative and/or real-time and/or in a multiplex format.
In an embodiment, a method employs TaqMan.RTM.-style probes
(dual-labeled probes to fluoresce upon 5'.fwdarw.3' exonuclease
activity). In an embodiment, a method employs TaqMan.RTM.-style
probes and oligonucleotides that selectively hybridize to the Rf4
gene. In an embodiment, the Rf4 gene probes can be coupled to a
detectable label (e.g., 6-carboxyfluorescein) at the 5' end of the
oligonucleotide. In an embodiment, the oligonucleotide can also be
coupled to a quencher moiety at the 3' end. An example of a
quencher moiety for the Rf4 gene probes is Black Hole Quencher.TM.
(Biosearch Technologies, Novato, Calif.). Suitable instrumentation
will thereby detect the fluorescence produced from the cleavage of
the oligonucleotide probe by the nuclease activity of the DNA
polymerase during replication. Analysis software then determines
the quantity of amplification product based upon the fluorescence
data.
[0040] In an embodiment, a method for determining zygosity of an
Rf4 gene in a corn plant includes a) performing a first PCR assay
using a first probe, a first forward primer, and a first reverse
primer on a polynucleotide sample from a corn plant; b) performing
a second PCR assay using a second probe, a second forward primer,
and a second reverse primer on the polynucleotide sample from a
corn plant; c) quantifying the first and second probes; and d)
comparing the quantified first and second probes to determine
zygosity. In an embodiment, the probes are detectably labeled. In
an embodiment, the primers and probes are specific for the Rf4 gene
in a corn plant. In an embodiment, a forward primer specific for
the Rf4 gene comprises SEQ ID NO:1, 11, or 15. In an embodiment, a
reverse primer specific for the Rf4 gene comprises SEQ ID NO:2, 14,
or 18. In an embodiment, a probe specific for the Rf4 gene
comprises SEQ ID NO:4, 13, or 17.
[0041] In an embodiment, a PCR assay method can include loading a
PCR reaction mixture in a PCR assay tube, wherein the PCR reaction
mixture comprises a polymerase with 5' to 3' nuclease activity,
deoxynucleotides, a buffer, a first and a second forward primer, a
first and a second reverse primer, a first and a second probe, and
a polynucleotide sample, and wherein the first probe and the second
probe comprise fluorescent dyes with distinguishable
excitation/emission spectra; and performing an amplification
step(s) under amplification conditions such that the 5' to 3'
nuclease activity of the polymerase cleaves the first and second
probes, thereby releasing fluorescent dyes comprising
distinguishable excitation/emission spectra.
[0042] In another embodiment, a PCR assay method to determine the
zygosity of the Rf4 gene includes loading a PCR reaction mixture in
a PCR assay tube, wherein the PCR reaction mixture comprises a
polymerase with 5' to 3' nuclease activity, deoxynucleotides, a
buffer, a first and/or a second forward primer, a first and/or a
second reverse primer, a first and/or a second probe, and a
polynucleotide sample; and performing an amplification step(s)
under conditions such that the 5' to 3' nuclease activity of the
polymerase cleaves the first or second probe, thereby releasing
fluorescent dyes comprising distinguishable excitation/emission
spectra.
[0043] In some embodiments, a label comprises a fluorescent dye
(e.g., a rhodamine dye (e.g., R6G, R110, TAMRA, ROX, etc.), a
fluorescein dye (e.g., JOE, VIC, TET, HEX, FAM, etc.), a
halofluorescein dye, a cyanine dye (e.g., CY3, CY3.5, CY5, CY5.5,
etc.), a Bodipy.RTM. dye (e.g., FL, 530/550, TR, TMR, etc.), an
Alexa Fluor.RTM. dye (e.g., 488, 532, 546, 568, 594, 555, 653, 647,
660, 680, etc.), a dichlororhodamine dye, an energy transfer dye
(e.g., Bigdye.RTM.. v 1 dyes, Bigdye.RTM. v 2 dyes, Bigdye.RTM. v 3
dyes, etc.), Lucifer dyes (e.g., Lucifer yellow, etc.), Cascade
Blue.RTM., Oregon Green.RTM., and the like. Fluorescent dyes can be
distinguished and measured during amplification by their emitted
excitation and/or emission spectra.
[0044] Examples of quenchers include, but are not limited to, Black
Hole Quencher.TM. 1 (BHQ1; Biosearch Technologies, Novato, Calif.),
Iowa Black.TM. (Integrated DNA Technologies), Dabcyl, QSY-7,
AbsoluteQuencher, Eclipse.RTM., and Minor Groove Binder (MGB)
quencher (Nanogen Inc., San Diego, Calif.).
EXAMPLES
[0045] Due to the practical importance of cytoplasmic male
sterility and pollen fertility restoration in maize hybrid seed
production and the necessity of determining the restoration
function of finished lines in the germplasm pool, a high throughput
endpoint TaqMan.RTM. PCR based zygosity assay was developed to
detect and test the zygosity status at Rf4 gene locus efficiently
and specifically.
Materials and Methods
[0046] Plant Genetic Material.
[0047] Nine maize inbred lines were used to develop an endpoint
TaqMan.RTM. zygosity assay, including three Rf4 fertility
restoration lines, three non-restoration lines for CMS C-type, two
CMS C-type lines, and one non-restorer and non-CMS-C line. Seeds
and/or leaf tissues were sampled for genomic DNA extraction. After
the genomic DNA was extracted, the DNA from an Rf4 fertility
restoration line was mixed in equal parts with the DNA from a
non-restoration CMS C-type line.
[0048] One F2 mapping population from the cross of an Rf4 fertility
restoration line was mixed in equal parts with the DNA from a
non-restoration CMS C-type line resulted in the production with 500
individual plant lines. Samples from this cross were used to
validate the endpoint TaqMan.RTM. zygosity assay. This population
was created in Mexico winter nursery in the end of 2009. Leaf
tissues were sampled for DNA extraction.
[0049] DNA Extraction from Seeds.
[0050] Needle-nosed pliers were used to pull the embryo out of a
corn kernel and put into a 1.2 mL sample tube (8 kernels from each
line). The pliers were wiped clean in-between kernels. One tungsten
alloy bead (.about.1/8 inch diameter) was added to each tube. Using
a Qiagen.TM. DNA isolation kit, 350 .mu.l of 65.degree. C. AP1
working solution containing 1 .mu.L RNase and 1 .mu.L Reagent DX
was added to each tube. Then each tube was capped and ground at
1,500 strokes per minute in an SPEX 2000 Geno/Grinder.RTM. (SPEX
SamplePrep LLC, Metuchen, N.J.). Subsequently, each tube was spun
for 10 seconds at 1500 RPM to remove liquid from the caps. The caps
were removed, and 114 .mu.L of AP2 was added. The tubes were capped
again and hand-shaken for 15 seconds. Samples were incubated for 10
minutes at -20.degree. C. The samples were centrifuged at 6,000 RPM
for 5 minutes. Then each tube was uncapped and 360 .mu.L of
supernatant was transferred to tubes containing 540 .mu.L of AP3/E
(200 proof ethanol already added to AP3/E). The tubes were capped
and hand-shaken for 15 seconds. Then the tubes were centrifuged for
10 seconds at 1500 RPM to remove liquid from the caps. 900 .mu.L
was transferred to a DNeasy.RTM. filter plate (Qiagen, Valencia,
Calif.). The filter plates were centrifuged on an S-Block for 4
minutes at 6000 RPM. Flow-through was poured out from the S-Block
and 800 .mu.L of AW (wash buffer--200 proof ethanol already added
to AW) was added to each well of the filter plate. The filter
plates were centrifuged on an S-Block for 4 minutes at 6000 RPM.
200 .mu.L of 200 proof ethanol were added to each well and
centrifuged on the S-Block for 1 minute. The filter plates were
placed onto a clean, dry S-block and centrifuged for 15 minutes to
dry filters. The filter plates were then placed onto a clean rack
of tubes in correct orientation. 100 .mu.L of AE was added to each
well twice. Each time, the AE was incubated for 1 minute at room
temperature and centrifuged for 2 minutes at 6000 RPM. The filter
plates were removed and tubes capped that contained DNA. The
extracted DNA was stored at 4.degree. C.
[0051] DNA Extraction from Leaf.
[0052] Leaf punches (8/plant) were collected from one-month old
seedlings, and DNA was extracted using a Biocel.RTM. 1800 (Agilent
Inc., Santa Clara, Calif.). Specifically, one tungsten alloy bead
(.about.1/8 inch diameter) was added to each tube. Then 300 .mu.L
of RLT Lysis Buffer was added to each tube. The tubes were capped
and ground for 6 minutes at 1500 strokes per minute in a SPEX 2000
Geno/Grinder.RTM. (SPEX SamplePrep LLC). Then the samples were
centrifuged 6000 RPM for 5 minutes. The tubes were uncapped.
[0053] The following steps were then performed using a Biocel.RTM.
1800. 200 .mu.L of supernatant was transferred to a 1.1 mL square
well round bottom assay plate containing 10 .mu.L MagAttract.RTM.
Suspension G Bead (Qiagen) and incubated for 2 minutes. Each well
was shaken at 1200 RPM for 40 seconds and then incubated for 2
minutes. The assay plates were placed onto a magnet shelf and the
beads were allowed to separate for 40 seconds. Supernatant was then
removed. The first time washing, 190 .mu.L RPW wash buffer
(premixed RNase.RTM. and isopropanol to RPW) was addend and shaken
at 1200 RPM for 40 seconds. The assay plate was again placed onto a
magnet shelf and the beads were allowed to separate for 20 seconds.
The supernatant was removed. The second time washing, 190 .mu.L of
100% ethanol wash buffer was added and shaken at 1200 RPM for 40
seconds. The assay plate was again placed onto a magnet shelf, and
the beads were allowed to separate for 20 seconds. The third time
washing, 190 .mu.L of 100% ethanol wash buffer was added and shaken
at 1200 RPM for 40 seconds. The assay plates were placed onto a
magnet shelf and the beads were allowed to separate for 20 seconds.
The supernatant was removed, and the plate incubated for 5 minutes
at room temperature. 100 .mu.L of AE elution buffer was added and
shaken for 2 minutes. The assay plates were placed onto a magnet
shelf and the beads were allowed to separate for 30 seconds. The
supernatant was transferred to clean, sealed plates and DNA was
stored at 4.degree. C. DNA was quantified by using PicoGreen.RTM.
(Molecular Probes Inc., Eugene, Oreg.) and normalized to 10
ng/.mu.L for further application.
[0054] TaqMan.RTM. PCR Assay Design and Validation.
[0055] Primer Express.RTM. 3.0 (Perkin-Elmer Corp., Foster City,
Calif.) was utilized to design TaqMan.RTM. assay primers and probes
(Table 1). DNA sequences from Rf4 restoration lines, CMS-C lines,
non-CMS-C line and corn line B73 were used. Primers CMSCF and CMSCR
were designed for both Rf4 and CMS-C/non-restorer. Probes RCMSC and
WCMSC were Rf4 specific at two nucleotides (TT) and
CMS-C/non-restorer specific at two nucleotides (AC),
respectively.
TABLE-US-00001 TABLE 1 Sequences of the primers and MGB probes for
Rf4 gene specific zygosity assay: SEQ CMS-C Rf4 TagMan .RTM.
primers and Primer ID probes name NO: Forward primer: CMSCF 1 5'-
CAACGGCGTCGAGAAGAAG -3' Reverse primer: CMSCR 2 5'-
TAACGTTGGGTATGAGGTGCAT-3' rf4(non-restorer) probe: WCMSC 3 5'-
ACCGAGAAGTACACCGC- 3' Rf4 (restorer) probe: RCMSC 4 5'-
CACCGAGAAGTTTACGGC- 3'
[0056] Primers and probes with FAM or VIC and Minor Grove Binding
Non Flourescence Quencher I (MGBFQ) dyes were synthesized by
Applied Biosystems (Foster City, Calif.), and were dissolved in
1.times. Tris-EDTA buffer to 100 .mu.M concentration. TaqMan.RTM.
gene expression master mixes (Applied Biosystems; and Catalog
#4370048) were used for all the PCR reactions.
[0057] Real time PCR reactions in 10 .mu.L volume were set up
according to Table 2 using 384-well plate on 7900HT Fast Real-Time
PCR System (Applied Biosystems) starting with 50.degree. C. for 2
minutes, then denaturing at 95.degree. C. for 10 minutes, followed
by 50 cycles of 95.degree. C. for 15 seconds, 60.degree. C. for 1
minute. Fluorescence signals were recorded at the end of each
cycle.
TABLE-US-00002 TABLE 2 Real-Time PCR Components Reagents Working
Con. Required Con. 1x vol. (.mu.L) PVP 0.5% 0.08% 1.55 Gene
Expression MM 2X 1X 6.55 Primer Mix 20 .mu.M 0.5 .mu.M 0.25
RCMSC_FAMprobe 20 .mu.M 0.2 .mu.M 0.1 WCMSC_VICprobe 20 .mu.M 0.2
.mu.M 0.1 Total Mix Vol. (.mu.L) -- -- 7 DNA (10 ng/ul) -- -- 3
Final PCR vol. (.mu.L) -- -- 10
End-point TaqMan.RTM. PCR assays in 10 .mu.L volume was also set up
according to Table 2 using 384-well plates. ABI GeneAmp.RTM. PCR
System 9700 (Applied Biosystems, Foster City, Calif.) was used for
amplification starting with 50.degree. C. for 2 minutes, then
denaturing at 95.degree. C. for 15 minutes, followed by 40 cycles
of 92.degree. C. for 15 seconds and 60.degree. C. for 1 minute. PCR
products were measured using Synergy GenS Microplate Reader
(BioTek, Winooski, Vt.) and Kraken KLIMS system (KBioscience,
England). The instrument settings of recommended wavelengths for
reading the PCR results are listed in Table 3.
TABLE-US-00003 TABLE 3 Instrument settings with recommended
wavelengths for reading the PCR products: Dye Excitation (nm)
Emission (nm) FAM (Rf4) 485 535 VIC (CMS-C or non-restorer) 485
560
[0058] Data Analysis.
[0059] For real-time PCR, the SDS RQ Manager Software performed
analyses of relative quantitation data generated by the Applied
Biosystems 7900HT Fast Real-Time PCR System. This software displays
the amplification plot data in a logarithmic plot of
baseline-corrected normalized reporter signal vs. cycle number. The
plot displays the amplification curve for each cell selected within
the plate grid.
[0060] Following the completion of the endpoint TaqMan.RTM. PCR and
fluorescence reading, the raw fluorescence intensity data directly
from the plate reader were analyzed in the KLIMS system. A graph
with RFU (relative fluorescence unit) of FAM as x-axis and VIC as
y-axis were generated. Determinations of zygosity were made based
on the cluster separation in a cluster view.
Results and Discussion
[0061] Maize CMS-C Rf4 sequence. High quality Rf4 gene sequences
were obtained through cloning and sequencing from six lines (three
Rf4 restoration lines: SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:9;
one CMS-C line: SEQ ID NO:6; one non-CMS-C line: SEQ ID NO:7; and,
the B73 line: SEQ ID NO:5). Sequences were analyzed and aligned
using Sequencher.RTM. 4.8 (Gene Codes Corp., Ann Arbor, Mich.).
Rf4-bHLH genomic sequence alignment is depicted below.
TABLE-US-00004 SEQ ID NO. 5 (1)
GGCAAGCTAATGGGGTACATATGGAAGGAGGAAACCAAGTCGATCGTCGTCGTAGCATGTCGGTGTG-
GGTACTACA CTACACACACATATACATGGGCAA SEQ ID NO: 6 (1)
GGCAAGCTAATGGGGTACATATGGAAGGAGGAAACCAAGTCGATCGTCGTCGTAGCATGTCGGTGTG-
GGTACTACA CTACACACACATATACATGGGCAA SEQ ID NO: 7 (1)
GGCAAGCTAATGGGGTACATATGGAAGGAGGAAACCAAGTCGATCGTCGTCGTAGCATGTCGGTGTG-
GGTACTACA CTACACACACATATACATGGGCAA SEQ ID NO: 8 (1)
GGCAAGCTAATGGGGTACATATGGAAGGAGGAAACCAAGTCGATCGTCGTCGTAGCATGTCGGTGTG-
GGTACTACA CTACACACACATATACATGGGCAA SEQ ID NO: 9 (1)
GGCAAGCTAATGGGGTACATATGGAAGGAGGAAACCAAGTCGATCGTCGTCGTAGCATGTCGGTGTG-
GGTACTACA CTACACACACATATACATGGGCAA SEQ ID NO: 10 (1)
GGCAAGCTAATGGGGTACATATGGAAGGAGGAAACCAAGTCGATCGTCGTCGTAGCATGTCGGTGTG-
GGTACTACA CTACACACACATATACATGGGCAA (101)
CGCAAGGCCACCTTTCTGAATCCTGCATGAGCGTGTACCACTAGAATTGTCAGTGTGTGCGGTGTAT-
GGCAGGTTT TTGGTTCGGCAAGTGGGGCCCTCC (101)
CGCAAGGCCACCTTTCTGAATCCTGCATGAGCGTGTACCACTAGAATTGTCAGTGTGTGCGGTGTAT-
GGCAGGTTT TTGGTTCGGCAAGTGGGGCCCTCC (101)
CGCAAGGCCACCTTTCTGAATCCTGCATGAGCGTGTACCACTAGAATTGTCAGTGTGTGCGGTGTAT-
GGCAGGTTT TTGGTTCGGCAAGTGGGGCCCTCC (101)
CGCAAGGCCACCTTTCTGAATCCTGCATGAGCGTGTACCACTAGAATTGTCAGTGTGTGCGGTGTAT-
GGCAGGTTT TTGGTTCGGCAAGTGGGGCCCTCC (101)
CGCAAGGCCACCTTTCTGAATCCTGCATGAGCGTGTACCACTAGAATTGTCAGTGTGTGCGGTGTAT-
GGCAGGTTT TTGGTTCGGCAAGTGGGGCCCTCC (101)
CGCAAGGCCACCTTTCTGAATCCTGCATGAGCGTGTACCACTAGAATTGTCAGTGTGTGCGGTGTAT-
GGCAGGTTT TTGGTTCGGCAAGTGGGGCCCTCC (201)
GGGGAGGAATCTCAGTAACAAACCGCTCTTCTGAAAAGGTCAGCCATCCCCGGTCCGGTCCGGTGAT-
GTCGTCGCT GTCGCTCTGCTAGCTTGCTGCCGA (201)
GGGGAGGAATCTCAGTAACAAACCGCTCTTCTGAAAAGGTCAGCCATCCCCGGTCCGGTCCGGTGAT-
GTCGTCGCT GTCGCTCTGCTAGCTTGCTGCCGA (201)
GGGGAGGAATCTCAGTAACAAACCGCTCTTCTGAAAAGGTCAGCCATCCCCGGTCCGGTCCGGTGAT-
GTCGTCGCT GTCGCTCTGCTAGCTTGCTGCCGA (201)
GGGGAGGAATCTCAGTAACAAACCGCTCTTCTGAAAAGGTCAGCCATCCCCGGTCCGGTCCGGTGAT-
GTCGTCGCT GTCGCTCTGCTAGCTTGCTGCCGA (201)
GGGGAGGAATCTCAGTAACAAACCGCTCTTCTGAAAAGGTCAGCCATCCCCGGTCCGGTCCGGTGAT-
GTCGTCGCT GTCGCTCTGCTAGCTTGCTGCCGA (201)
GGGGAGGAATCTCAGTAACAAACCGCTCTTCTGAAAAGGTCAGCCATCCCCGGTCCGGTCCGGTGAT-
GTCGTCGCT GTCGCTCTGCTAGCTTGCTGCCGA (301)
TCCCCCCCCCCCCCCCCCCCCTTCTTCTCTCTACCCCTCCCTCCACCTCATAAATACTTAGTTTAAT-
AACCTTGCA CTGCCGCAGTAGCCCTTAACTGCT (301)
TCCCCCCCCCCCCCCCCCCCCTTCTTCTCTCTACCCCTCCCTCCACCTCATAAATACTTAGTTTAAT-
AACCTTGCA CTGCCGCAGTAGCCCTTAACTGCT (301)
TCCCCCCCCC-----------TTCTTCTCTCTACCCCTCCCTCCACCTCATAAATACTTAGTTTAAT-
AACCTTGCA CTGCCGCAGTAGCCCTTAACTGCT (301)
TCCCCCCCCCCCCCCCCCCCCTTCTTCTCTCTACCCCTCCCTCCACCTCATAAATACTTAGTTTAAT-
AACCTTGCA CTGCCGCAGTAGCCCTTAACTGCT (301)
TCCCCCCCCCCCCCCCCCCCCTTCTTCTCTCTACCCCTCCCTCCACCTCATAAATACTTAGTTTAAT-
AACCTTGCA CTGCCGCAGTAGCCCTTAACTGCT (301)
TCCCCCCCCCCCCCCCCCCCCTTCTTCTCTCTACCCCTCCCTCCACCTCATAAATACTTAGTTTAAT-
AACCTTGCA CTGCCGCAGTAGCCCTTAACTGCT (401)
GCTATCTATCTCTTTTCTGAAGGAAAAAAAAGGTTTGATACTCCTCTACCTAGCTAGTCCTGCATGC-
CGCTAATGT GCGTCTTGCCTGTTTATTTGTTCT (401)
GCTATCTATCTCTTTTCTGAAGGAAAAAAAAGGTTTGATACTCCTCTACCTAGCTAGTCCTGCATGC-
CGCTAATGT GCGTCTTGCCTGTTTATTTGTTCT (390)
GCTATCTATCTCTTTTCTGAAGGAAAAAAAAGGTTTGATACTCCTCTACCTAGCTAGTCCTGCATGC-
CGCTAATGT GCGTCTTGCCTGTTTATTTGTTCT (401)
GCTATCTATCTCTTTTCTGAAGGAAAAAAAAGGTTTGATACTCCTCTACCTAGCTAGTCCTGCATGC-
CGCTAATGT GCGTCTTGCCTGTTTATTTGTTCT (401)
GCTATCTATCTCTTTTCTGAAGGAAAAAAAAGGTTTGATACTCCTCTACCTAGCTAGTCCTGCATGC-
CGCTAATGT GCGTCTTGCCTGTTTATTTGTTCT (401)
GCTATCTATCTCTTTTCTGAAGGAAAAAAAAGGTTTGATACTCCTCTACCTAGCTAGTCCTGCATGC-
CGCTAATGT GCGTCTTGCCTGTTTATTTGTTCT DAS-CMS21 (501)
TAATAAGGGCTGCCTATCTATTATATTTTGCACCTGTTTTGCTGTGTTCTTGGTAACTAGCTTAATT-
CCTTCGCCT ACAATCGTCAAATCCCCCCCATCA (501)
TAATAAGGGCTGCCTATCTATTATATTTTGCACCTGTTTTGCTGTGTTCTTGGTAACTAGCTTAATT-
CCTTCGCCT ACAATCGTCAAATCCCCCCCATCA (490)
TAATAAGGGCTGCCTATCTATTATATTTTGCACCTGTTTTGCTGTGTTCTTGGTAACTAGCTTAATT-
CCTTTGCCT ACAATCGTCAAATCCCCCCCATCA (501)
TAATAAGGGCTGCCTATCTATTATATTTTGCACCTGTTTTGCTGTGTTCTTGGTAACTAGCTTAATT-
CCTTTGCCT ACAATCGTCAAATCCCCCCCATCA (501)
TAATAAGGGCTGCCTATCTATTATATTTTGCACCTGTTTTGCTGTGTTCTTGGTAACTAGCTTAATT-
CCTTTGCCT ACAATCGTCAAATCCCCCCCATCA (501)
TAATAAGGGCTGCCTATCTATTATATTTTGCACCTGTTTTGCTGTGTTCTTGGTAACTAGCTTAATT-
CCTTTGCCT ACAATCGTCAAATCCCCCCCATCA (601)
TCAGTCAGATGAACTTTTGATCGAATTGAAGTTGTTCTTCTAATTCGGCCCCAGCAGCGCCCATGCA-
TCTGGTTTT ATTTGCTTTCTGTTGGGTATAATA (601)
TCAGTCAGATGAACTTTTGATCGAATTGAAGTTGTTCTTCTAATTCGGCCCCAGCAGCGCCCATGCA-
TCTGGTTTT ATTTGCTTTCTGTTGGGTATAATA (590)
TCAGTCAGATGAACTTTTGATCGAATTGAAGTTGTTCTTCTAATTCGGCCCCAGCAGCGCCCATGCA-
TCTGGTTTT ATTTGCTTTCTGTTGGGTATAATA (601)
TCAGTCAGATGAACTTTTGATCGAATTGAAGTTGTTCTTCTAATTCGGCCCCAGCAGCGCCCATGCA-
TCTGGTTTT ATTTGCTTTCTGTTGGGTATAATA (601)
TCAGTCAGATGAACTTTTGATCGAATTGAAGTTGTTCTTCTAATTCGGCCCCAGCAGCGCCCATGCA-
TCTGGTTTT ATTTGCTTTCTGTTGGGTATAATA (601)
TCAGTCAGATGAACTTTTGATCGAATTGAAGTTGTTCTTCTAATTCGGCCCCAGCAGCGCCCATGCA-
TCTGGTTTT ATTTGCTTTCTGTTGGGTATAATA (701)
TGCAAGACCTTTTGTTGCTAGGGCAAGGCTGCAACCACATGCGTGTACTGAACTCATGATGTAACTC-
ATCCTTTTT GTTTGCTCACAGAATCACTACTCT (701)
TGCAAGACCTTTTGTTGCTAGGGCAAGGCTGCAACCACATGCGTGTACTGAACTCATGATGTAACTC-
ATCCTTTTT GTTTGCTCACAGAATCACTACTCT (690)
TGCAAGACCTTTTGTTGCTAGGGCAAGGCTGCAACCACATGCGTGTACTGAACTCATGATGTAACTC-
ATCCTTTTT GTTTGCTCACAGAATCACTACTCT (701)
TGCAAGACCTTTTGTTGCTAGGGCAAGGCTGCAACCACATGCGTGTACTGAACTCATGATGTAACTC-
ATCCTTTTT GTTTGCTCACAGAATCACTACTCT (701)
TGCAAGACCTTTTGTTGCTAGGGCAAGGCTGCAACCACATGCGTGTACTGAACTCATGATGTAACTC-
ATCCTTTTT GTTTGCTCACAGAATCACTACTCT (701)
TGCAAGACCTTTTGTTGCTAGGGCAAGGCTGCAACCACATGCGTGTACTGAACTCATGATGTAACTC-
ATCCTTTTT GTTTGCTCACAGAATCACTACTCT (801)
ACTGCACTTCCTTTTCATCCGATCCGCAATCTTTTTTTTCTTTTACATGCTTTAGTTTTCTCTCTTT-
CTTGATTAC AAACATGATTACTGGAACTTTCTT (801)
ACTGCACTTCCTTTTCATCCGATCCGCAATCTTTTTTTTCTTTTACATGCTTTAGTTTTCTCTCTTT-
CTTGATTAC AAACATGATTACTGGAACTTTCTT (790)
ACTGCACTTCCTTTTCATCCGATCCGCAATCTTTTTTTTCTTTTACATGCTTTAGTTTTCTCTCTTT-
CTTGATTAC AAACATGATTACTGGAACTTTCTT (801)
ACTGCACTTCCTTTTCATCCGATCCGCAATCTTTTTTTTCTTTTACATGCTTTAGTTTTCTCTCTTT-
CTTGATTAC AAACATGATTACTGGAACTTTCTT (801)
ACTGCACTTCCTTTTCATCCGATCCGCAATCTTTTTTTTCTTTTACATGCTTTAGTTTTCTCTCTTT-
CTTGATTAC AAACATGATTACTGGAACTTTCTT (801)
ACTGCACTTCCTTTTCATCCGATCCGCAATCTTTTTTTTCTTTTACATGCTTTAGTTTTCTCTCTTT-
CTTGATTAC AAACATGATTACTGGAACTTTCTT (901)
AGGCTGCCTTCCCCTTCCTTGGATCTGCTTTAGTTTTCTTTTTTGGGCTACCGCGCGCGGCTTATTT-
GAGTTTATC ACTTGCTGCATATACATAATATAT (901)
AGGCTGCCTTCCCCTTCCTTGGATCTGCTTTAGTTTTCTTTTTTGGGCTACCGCGCGCGGCTTATTT-
GAGTTTATC ACTTGCTGCATATACATAATATAT (890)
AGGCTGCCTTCCCCTTCCTTGGATCTGCTTTAGTTTTCTTTTTTGGGCTACCGCGCGCGGCTTATTT-
GAGTTTATC ACTTGCTGCATATACATAATATAT (901)
AGGCTGCCTTCCCCTTCCTTGGATCTGCTTTAGTTTTCTTTTTTGGGCTACCGCGCGCGGCTTATTT-
GAGTTTATC ACTTGCTGCATATACATAATATAT (901)
AGGCTGCCTTCCCCTTCCTTGGATCTGCTTTAGTTTTCTTTTTTGGGCTACCGCGCGCGGCTTATTT-
GAGTTTATC ACTTGCTGCATATACATAATATAT (901)
AGGCTGCCTTCCCCTTCCTTGGATCTGCTTTAGTTTTCTTTTTTGGGCTACCGCGCGCGGCTTATTT-
GAGTTTATC ACTTGCTGCATATACATAATATAT (1001)
ATATACATGCATGCGATGGCGTTCATGTTACTCAACTACAGATCTGTTTCTGTTCGTGTGTTTCAGT-
TCAGCGCGC
AGTTAAGCATAGCAGGACGACCAC (1001)
ATATACATGCATGCGATGGCGTTCATGTTACTCAACTACAGATCTGTTTCTGTTCGTGTGTTTCAGT-
TCAGCGCGC AGTTAAGCATAGCAGGACGACCAC (990)
ATATACATGCATGCGATGGCGTTCATGTTACTCAACTACAGATCTGTTTCTGTTCGTGTGTTTCAGT-
TCAGCGCGC AGTTAAGCATAGCAGGACGACCAC (1001)
ATATACATGCATGCGATGGCGTTCATGTTACTCAACTACAGATCTGTTTCTGTTCGTGTGTTTCAGT-
TCAGCGCGC AGTTAAGCATAGCAGGACGACCAC (1001)
ATATACATGCATGCGATGGCGTTCATGTTACTCAACTACAGATCTGTTTCTGTTCGTGTGTTTCAGT-
TCAGCGCGC AGTTAAGCATAGCAGGACGACCAC (1001)
ATATACATGCATGCGATGGCGTTCATGTTACTCAACTACAGATCTGTTTCTGTTCGTGTGTTTCAGT-
TCAGCGCGC AGTTAAGCATAGCAGGACGACCAC START (1101)
GACGATGTATCACCCGCAGTGCGAGCTCCTGACGATGGCGCACGAAACGCCGGACCTGGACGCCGGC-
CAGCCGCAC CTAACCGTCTCCGGCGTCGCCAGC (1101)
GACGATGTATCACCCGCAGTGCGAGCTCCTGACGATGGCGCACGAAACGCCGGACCTGGACGCCGGC-
CAGCCGCAC CTAACCGTCTCCGGCGTCGCCAGC (1090)
GACGATGTATCACCCGCAGTGCGAGCTCCTGACGATGGCGCACGAAACGCCGGACCTGGACGCCGGC-
CAGCCGCAC CTAACCGTCTCCGGCGTCGCCAGC (1101)
GACGATGTATCACCCGCAGTGCGAGCTCCTGACGATGGCGCACGAAACGCCGGACCTGGACGCCGGC-
CAGCCGCAC CTAACCGTCTCCGGCGTCGCCAGC (1101)
GACGATGTATCACCCGCAGTGCGAGCTCCTGACGATGGCGCACGAAACGCCGGACCTGGACGCCGGC-
CAGCCGCAC CTAACCGTCTCCGGCGTCGCCAGC (1101)
GACGATGTATCACCCGCAGTGCGAGCTCCTGACGATGGCGCACGAAACGCCGGACCTGGACGCCGGC-
CAGCCGCAC CTAACCGTCTCCGGCGTCGCCAGC (1201)
ATCCCGGCAGAGCTGAGCTTCCACCTGCTGCACTCGCTCGACGCCGCGGCGGCGGTCAATCCCGTCA-
CGGCGCCGC CGCAGTCCACCATCGACTACTTCC (1201)
ATCCCGGCAGAGCTGAGCTTCCACCTGCTGCACTCGCTCGACGCCGCGGCGGCGGTCAATCCCGTCA-
CGGCGCCGC CGCAGTCCACCATCGACTACTTCC (1190)
ATCCCGGCAGAGCTGAGCTTCCACCTGCTGCACTCGCTCGACGCCGCGGCGGCGGTCAATCCCGTCA-
CGGCGCCGC CGCAGTCCACCATCGACTACTTCC (1201)
ATCCCGGCAGAGCTGAGCTTCCACCTGCTGCACTCGCTCGACGCCGCGGCGGCGGTCAATCCCGTCA-
CGGCGCCGC CGCAGTCCACCATCGACTACTTCC (1201)
ATCCCGGCAGAGCTGAGCTTCCACCTGCTGCACTCGCTCGACGCCGCGGCGGCGGTCAATCCCGTCA-
CGGCGCCGC CGCAGTCCACCATCGACTACTTCC (1201)
ATCCCGGCAGAGCTGAGCTTCCACCTGCTGCACTCGCTCGACGCCGCGGCGGCGGTCAATCCCGTCA-
CGGCGCCGC CGCAGTCCACCATCGACTACTTCC (1301)
TCGGCGGCGCCGATCCCCACCAGCAGGCCATGCAGTACGAGCCGCTGCCGCCCGCCGCGGGCGGCCA-
CCACCAGTA CACCATGGACATGTTCCGCGACTA (1301)
TCGGCGGCGCCGATCCCCACCAGCAGGCCATGCAGTACGAGCCGCTGCCGCCCGCCGCGGGCGGCCA-
CCACCAGTA CACCATGGACATGTTCCGCGACTA (1290)
TCGGCGGCGCCGATCCCCACCAGCAGGCCATGCAGTACGAGCCGCTGCCGCCCGCCGCGGGCGGCCA-
CCACCAGTA CACCATGGACATGTTCCGCGACTA (1301)
TCGGCGGCGCCGATCCCCACCAGCAGGCCATGCAGTACGAGCCGCTGCCGCCCGCCGCGGGCGGCCA-
CCACCAGTA CACCATGGACATGTTCCGCGACTA (1301)
TCGGCGGCGCCGATCCCCACCAGCAGGCCATGCAGTACGAGCCGCTGCCGCCCGCCGCGGGCGGCCA-
CCACCAGTA CACCATGGACATGTTCCGCGACTA (1301)
TCGGCGGCGCCGATCCCCACCAGCAGGCCATGCAGTACGAGCCGCTGCCGCCCGCCGCGGGCGGCCA-
CCACCAGTA CACCATGGACATGTTCCGCGACTA DAS-CMS22(H/N) (1401)
CTGCGACGGCCACTACCCCACCGCCGAGCCGTACATCCGCGGGACAATGACTGGAGCCCTCGTGTTC-
GGGGCCACC GACGACGACGACTCGGCCGCTGCC (1401)
CTGCGACGGCCACTACCCCACCGCCGAGCCGTACATCCGCGGGACAATGACTGGAGCCCTCGTGTTC-
GGGGCCACC GACGACGACGACTCGGCCGCTGCC (1390)
CTGCGACGGCCACTACCCCACCGCCGAGCCGTACATCCGCGGGACAATGACTGGAGCCCTCGTGTTC-
GGGGCCACC GACGACGACGACTCGGCCGCTGCC (1401)
CTGCGACGGCAACTACCCCACCGCCGAGCCGTACATCCGCGGGACAATGACTGGAGCCCTCGTGTTC-
GGGGCCACC GACGACGACGACTCGGCCGCTGCC (1401)
CTGCGACGGCAACTACCCCACCGCCGAGCCGTACATCCGCGGGACAATGACTGGAGCCCTCGTGTTC-
GGGGCCACC GACGACGACGACTCGGCCGCTGCC (1401)
CTGCGACGGCAACTACCCCACCGCCGAGCCGTACATCCGCGGGACAATGACTGGAGCCCTCGTGTTC-
GGGGCCACC GACGACGACGACTCGGCCGCTGCC DAS-CMS23(-/A) (1500)
---TACATGCCCGGGGGGCACTTTGAGACCTCCCCGCCGCCGCCACGCGCCACCGGCCGCGGCAGGA-
AGCGGGGCA GGGCGCTGGGCGGCGGCTTCCATG (1500)
---TACATGCCCGGGGGGCACTTTGAGACCTCCCCGCCGCCGCCACGCGCCACCGGCCGCGGCAGGA-
AGCGGGGCA GGGCGCTGGGCGGCGGCTTCCATG (1489)
---TACATGCCCGGGGGGCACTTTGAGACCTCCCCGCCGCCGCCACGCGCCACCGGCCGCGGCAGGA-
AGCGGGGCA GGGCGCTGGGCGGCGGCTTCCATG (1501)
GCCTACATGCCCGGGGGGCACTTTGAGACCTCCCCGCCGCCGCCACGCGCCACCGGCCGCGGCAGGA-
AGCGGGGCA GGGCGCTGGGCGGCGGCTTCCATG (1501)
GCCTACATGCCCGGGGGGCACTTTGAGACCTCCCCGCCGCCGCCACGCGCCACCGGCCGCGGCAGGA-
AGCGGGGCA GGGCGCTGGGCGGCGGCTTCCATG (1501)
GCCTACATGCCCGGGGGGCACTTTGAGACCTCCCCGCCGCCGCCACGCGCCACCGGCCGCGGCAGGA-
AGCGGGGCA GGGCGCTGGGCGGCGGCTTCCATG DAS-CMS35(Y/F) DAS-CMS24 (1598)
CTGTGCTGGCCAACGGCGTCGAGAAGAAGGAGAAGCAGCGCCGGCTGCGGCTCACCGAGAAGTACAC-
CGCCCTCAT DAS-CMS25 GCACCTCATACCCAACGTTACAAA (1598)
CTGTGCTGGCCAACGGCGTCGAGAAGAAGGAGAAGCAGCGCCGGCTGCGGCTCACCGAGAAGTACAC-
CGCCCTCAT GCACCTCATACCCAACGTTACAAA (1587)
CTGTGCTGGCCAACGGCGTCGAGAAGAAGGAGAAGCAGCGCCGGCTGCGGCTCACCGAGAAGTACAC-
CGCCCTCAT GCACCTCATACCCAACGTTACAAA (1601)
CTGTGCTGGCCAACGGCGTCGAGAAGAAGGAGAAGCAGCGCCGGCTGCGGCTCACCGAGAAGTTTAC-
GGCCCTCAT GCACCTCATACCCAACGTTACGAA (1601)
CTGTGCTGGCCAACGGCGTCGAGAAGAAGGAGAAGCAGCGCCGGCTGCGGCTCACCGAGAAGTTTAC-
GGCCCTCAT GCACCTCATACCCAACGTTACGAA (1601)
CTGTGCTGGCCAACGGCGTCGAGAAGAAGGAGAAGCAGCGCCGGCTGCGGCTCACCGAGAAGTTTAC-
GGCCCTCAT GCACCTCATACCCAACGTTACGAA DAS-CMS26 (1698)
GGTCGTAC-------------------CAAATCCTCCTCTTATGTTCGTC---CATCGTTTCAAATT-
AAGTTAAAA AATTAATTCACGGTTCTTGTTGTT (1698)
GGTCGTAC-------------------CAAATCCTCCTCTTATGTTCGTC---CATCGTTTCAAATT-
AAGTTAAAA AATTAATTCACGGTTCTTGTTGTT (1687)
GGTCGTAC-------------------CAAATCCTCCTCTTATGTTCGTC---CATCGTTTGAAATT-
AAGTTAAAA AATTAATTCACGGTTCTTGTTGTT (1701)
GGTCGTACGGCGTACTTGCGCGCGGACCAAATCCTCCTCTTATGTTCGTCGTCCATCGTCTCAAATT-
AA------- ------TTCACGGTTCTTGTTGTT (1701)
GGTCGTACGGCGTACTTGCGCGCGGACCAAATCCTCCTCTTATGTTCGTCGTCCATCGTCTCAAATT-
AA------- ------TTCACGGTTCTTGTTGTT (1701)
GGTCGTACGGCGTACTTGCGCGCGGACCAAATCCTCCTCTTATGTTCGTCGTCCATCGTCTCAAATT-
AA------- ------TTCACGGTTCTTGTTGTT DAS-CMS27 (1776)
---TATTTTTTGCGCACTGCAGACTGATAGGGCGACGGTGATCTCGGACGCGATCGAGTACATCCAG-
GAGCTGGGG AGGACGGTGGAGGAGCTGACGCTG (1776)
---TATTTTTTGCGCACTGCAGACTGATAGGGCGACGGTGATCTCGGACGCGATCGAGTACATCCAG-
GAGCTGGGG AGGACGGTGGAGGAGCTGACGCTG (1765)
---TATTTTTTGCGCACTGCAGACTGATAGGGCGACGGTGATCTCGGACGCGATCGAGTACATCCAG-
GAGCTGGGG AGGACGGTGGAGGAGCTGACGCTG (1788)
GTTTATTTTTTGCGCACTGCAGACTGATAGGGCGACGGTGATCTCGGACGCGATCGAGTACATCCAG-
GAGCTGGGG AGGACGGTGGAGGAGCTGACGCTG (1788)
GTTTATTTTTTGCGCACTGCAGACTGATAGGGCGACGGTGATCTCGGACGCGATCGAGTACATCCAG-
GAGCTGGGG AGGACGGTGGAGGAGCTGACGCTG (1788)
GTTTATTTTTTGCGCACTGCAGACTGATAGGGCGACGGTGATCTCGGACGCGATCGAGTACATCCAG-
GAGCTGGGG AGGACGGTGGAGGAGCTGACGCTG (1873)
CTGGTGGAGAAGAAGCGGCGCCGGAGGGAGCTGCAGGGGGACGTCGTGGACGCGGCGCCGGCTGCGG-
TGGTTGCTG CCGCCGGTGAGGCGGAGAGCTCGG (1873)
CTGGTGGAGAAGAAGCGGCGCCGGAGGGAGCTGCAGGGGGACGTCGTGGACGCGGCGCCGGCTGCGG-
TGGTTGCTG CCGCCGGTGAGGCGGAGAGCTCGG (1862)
CTGGTGGAGAAGAAGCGGCGCCGGAGGGAGCTGCAGGGGGACGTCGTGGACGCGGCGCCGGCTGCGG-
TGGTTGCTG CCGCCGGTGAGGCGGAGAGCTCGG (1888)
CTGGTGGAGAAGAAGCGGCGCCGGAGGGAGCTGCAGGGGGACGTCGTGGACGCGGCGCCGGCTGCGG-
TGGTTGCTG CCGCCGGTGAGGCGGAGAGCTCGG (1888)
CTGGTGGAGAAGAAGCGGCGCCGGAGGGAGCTGCAGGGGGACGTCGTGGACGCGGCGCCGGCTGCGG-
TGGTTGCTG CCGCCGGTGAGGCGGAGAGCTCGG (1888)
CTGGTGGAGAAGAAGCGGCGCCGGAGGGAGCTGCAGGGGGACGTCGTGGACGCGGCGCCGGCTGCGG-
TGGTTGCTG CCGCCGGTGAGGCGGAGAGCTCGG DAS-CMS28(P/L) (1973)
AGGGCGAGGTGGCTCCTCCGCCGCCGGCCGTGCCGCGGCAGCCGATCCGGAGCACGTACATCCAGCG-
GCGGAGCAA GGACACGTCCGTGGACGTGCGGAT
(1973)
AGGGCGAGGTGGCTCCTCCGCCGCCGGCCGTGCCGCGGCAGCCGATCCGGAGCACGTACATCCAGCG-
GCGGAGCAA GGACACGTCCGTGGACGTGCGGAT (1962)
AGGGCGAGGTGGCTCCTCCGCCGCCGGCCGTGCCGCGGCAGCCGATCCGGAGCACGTACATCCAGCG-
GCGGAGCAA GGACACGTCCGTGGACGTGCGGAT (1988)
AGGGCGAGGTGGCTCCTCCGCCGCTGGCCGTGCCGCGGCAGCCGATCCGGAGCACGTACATCCAGCG-
GCGGAGCAA GGACACGTCCGTGGACGTGCGGAT (1988)
AGGGCGAGGTGGCTCCTCCGCCGCTGGCCGTGCCGCGGCAGCCGATCCGGAGCACGTACATCCAGCG-
GCGGAGCAA GGACACGTCCGTGGACGTGCGGAT (1988)
AGGGCGAGGTGGCTCCTCCGCCGCTGGCCGTGCCGCGGCAGCCGATCCGGAGCACGTACATCCAGCG-
GCGGAGCAA GGACACGTCCGTGGACGTGCGGAT (2073)
CGTGGAGGAGGACGTGAACATCAAGCTCACCAAGCGCCGGCGCGACGGGTGCCTCGCAGCCGCGTCG-
CGCGCGCTG DAS-CMS29 GATGACCTCCGCCTTGACCTCGTC (2073)
CGTGGAGGAGGACGTGAACATCAAGCTCACCAAGCGCCGGCGCGACGGGTGCCTCGCAGCCGCGTCG-
CGCGCGCTG GATGACCTCCGCCTTGACCTCGTC (2062)
CGTGGAGGAGGACGTGAACATCAAGCTCACCAAGCGCCGGCGCGACGGGTGCCTCGCAGCCGCGTCG-
CGCGCGCTG GATGACCTCCGCCTTGACCTCGTC (2088)
CGTGGAGGAGGACGTGAACATCAAGCTCACCAAGCGCCGGCGCGACGGGTGCCTCGCAGCCGCGTCG-
CGCGCGCTG GACGACCTCCGCCTTGACCTCGTC (2088)
CGTGGAGGAGGACGTGAACATCAAGCTCACCAAGCGCCGGCGCGACGGGTGCCTCGCAGCCGCGTCG-
CGCGCGCTG GACGACCTCCGCCTTGACCTCGTC (2088)
CGTGGAGGAGGACGTGAACATCAAGCTCACCAAGCGCCGGCGCGACGGGTGCCTCGCAGCCGCGTCG-
CGCGCGCTG GACGACCTCCGCCTTGACCTCGTC (2173)
CACCTCTCCGGCGGCAAGATCGGTGACTGTCAAATCTACATGTTCAACACCAAGGTACATACGAATA-
CGATACGTA GCCATTGATCGATCTGTAATTCTG (2173)
CACCTCTCCGGCGGCAAGATCGGTGACTGTCAAATCTACATGTTCAACACCAAGGTACATACGAATA-
CGATACGTA GCCATTGATCGATCTGTAATTCTG (2162)
CACCTCTCCGGCGGCAAGATCGGTGACTGTCAAATCTACATGTTCAACACCAAGGTACATACGAATA-
CGATACGTA GCCATTGATCGATCTGTAATTCTG (2188)
CACCTCTCCGGCGGCAAGATCGGTGACTGTCAAATCTACATGTTCAACACCAAGGTACATACGAATA-
CGATACGTA GCCATTGATCGATCTGTAATTCTG (2188)
CACCTCTCCGGCGGCAAGATCGGTGACTGTCAAATCTACATGTTCAACACCAAGGTACATACGAATA-
CGATACGTA GCCATTGATCGATCTGTAATTCTG (2188)
CACCTCTCCGGCGGCAAGATCGGTGACTGTCAAATCTACATGTTCAACACCAAGGTACATACGAATA-
CGATACGTA GCCATTGATCGATCTGTAATTCTG DAS-CMS30 (2273)
TAGCCTGACGATT---------------CCGAGGTTTCTG--------------------GTGCTAA-
AAAATGCAT CTTTTTTTCTCAGATGACAATGCT (2273)
TAGCCTGACGATT---------------CCGAGGTTTCTG--------------------GTGCTAA-
AAAATGCAT CTTTTTTTCTCAGATGACAATGCT (2262)
TAGCCTGACGATT---------------CCGAGGTTTCTG--------------------GTGCTAA-
AAAATGCAT CTTTTTTTCTCAGATGACAATGCT (2288)
TAGCCTGACGATTTCATGCATTACTTTTCCGAGGTTTCTGTGCTATACTACCTAACCTAGGTGCTAA-
AAAATGCAC CTTTTTTTCTCAGATGACAATGCT (2288)
TAGCCTGACGATTTCATGCATTACTTTTCCGAGGTTTCTGTGCTATACTACCTAACCTAGGTGCTAA-
AAAATGCAC CTTTTTTTCTCAGATGACAATGCT (2288)
TAGCCTGACGATTTCATGCATTACTTTTCCGAGGTTTCTGTGCTATACTACCTAACCTAGGTGCTAA-
AAAATGCAC CTTTTTTTCTCAGATGACAATGCT (2338)
TTCTGTCTTTGTTCACCGCAGATTCACAAGGGGTCTTCAGTGTTTGCGAGTGCAGTGGCCGGTAGGC-
TGATGGAAG STOP TGGTGGACGAGTACTAGGCTACCA (2338)
TTCTGTCTTTGTTCACCGCAGATTCACAAGGGGTCTTCAGTGTTTGCGAGTGCAGTGGCCGGTAGGC-
TGATGGAAG TGGTGGACGAGTACTAGGCTACCA (2327)
TTCTGTCTTTGTTCACCGCAGATTCACAAGGGGTCTTCAGTGTTTGCGAGTGCAGTGGCCGGTAGGC-
TGATGGAAG TGGTGGACGAGTACTAGGCTACCA (2388)
TTCTGTCTTTGTTCACCGCAGATTCACAAGGGGTCTTCAGTGTTTGCGAGTGCAGTGGCCGGTAGGC-
TGATGGAAG TGGTGGACGAGTACTAGGCTACCA (2388)
TTCTGTCTTTGTTCACCGCAGATTCACAAGGGGTCTTCAGTGTTTGCGAGTGCAGTGGCCGGTAGGC-
TGATGGAAG TGGTGGACGAGTACTAGGCTACCA (2388)
TTCTGTCTTTGTTCACCGCAGATTCACAAGGGGTCTTCAGTGTTTGCGAGTGCAGTGGCCGGTAGGC-
TGATGGAAG TGGTGGACGAGTACTAGGCTACCA DAS-CMS31 (2438)
TGCACTTGAATTTCTAGCTAGCTCTACGTACCGCGCTGCTATGAATCTAGCTATAGCGTTTCTTGGA-
TGAAAGACT AGTTAGTTGTTACCTTCTATCTTT (2438)
TGCACTTGAATTTCTAGCTAGCTCTACGTACCGCGCTGCTATGAATCTAGCTATAGCGTTTCTTGGA-
TGAAAGACT AGTTAGTTGTTACCTTCTATCTTT (2427)
TGCACTTGAATTTCTAGCTAGCTCTACGTACCGCGCTGCTATGAATCTAGCTATAGCGTTTCTTGGA-
TGAAAGACT AGTTAGTTGTTACCTTCTATCTTT (2488)
TGCACTTGAATTTCTAGCTAGCTCTACGTACCGCGCTGCTATGAATCTAGCTATAGCGTTTCTTGGA-
TGAAAGAAT AGTTAGTTGTTACCTTCTATCTTT (2488)
TGCACTTGAATTTCTAGCTAGCTCTACGTACCGCGCTGCTATGAATCTAGCTATAGCGTTTCTTGGA-
TGAAAGAAT AGTTAGTTGTTACCTTCTATCTTT (2488)
TGCACTTGAATTTCTAGCTAGCTCTACGTACCGCGCTGCTATGAATCTAGCTATAGCGTTTCTTGGA-
TGAAAGAAT AGTTAGTTGTTACCTTCTATCTTT (2538)
GCTTCAATTAAATCCGCTTGCTCGTTACAGACTGAGTTTGTTTCTAAATGTCAAGGTTGTTTTGGTC-
AAATTGAAT AAATTGGCACACTGGCCTGTGAGG (2538)
GCTTCAATTAAATCCGCTTGCTCGTTACAGACTGAGTTTGTTTCTAAATGTCAAGGTTGTTTTGGTC-
AAATTGAAT AAATTGGCACACTGGCCTGTGAGG (2527)
GCTTCAATTAAATCCGCTTGCTCGTTACAGACTGAGTTTGTTTCTAAATGTCAAGGTTGTTTTGGTC-
AAATTGAAT AAATTGGCACACTGGCCTGTGAGG (2588)
GCTTCAATTAAATCCGCTTGCTCGTTACAGACTGAGTTTGTTTCTAAATGTCAAGGTTGTTTTGGTC-
AAATTGAAT AAATTGGCACACTGGCCTGTGAGG (2588)
GCTTCAATTAAATCCGCTTGCTCGTTACAGACTGAGTTTGTTTCTAAATGTCAAGGTTGTTTTGGTC-
AAATTGAAT AAATTGGCACACTGGCCTGTGAGG (2588)
GCTTCAATTAAATCCGCTTGCTCGTTACAGACTGAGTTTGTTTCTAAATGTCAAGGTTGTTTTGGTC-
AAATTGAAT AAATTGGCACACTGGCCTGTGAGG (2638)
TTATTATATATATTTATGTGT-TTATTACTGGTCTATTAATTTGTCTTATTATTAATGTATTGCCTG-
TCAAGGAAT DAS-CMS32 AAATGGTATGATGACCATATTTAT (2638)
TTATTATATATATTTATGTGT-TTATTACTGGTCTATTAATTTGTCTTATTATTAATGTATTGCCTG-
TCAAGGAAT AAATGGTATGATGACCATATTTAT (2627)
TTATTATATATATTTATGTGT-TTATTACTGGTCTATTAATTTGTCCTATTATTAATGTATTGCCTG-
TCAAGGAAT AAATGATATGATGACCATATTTAT (2688)
TTATTATAT----TTATGTGTATTATTACTGGTCTATCAATTTGTCCTATTATT---GTATTGCCTG-
TCAAGGAAT AAATTGTATGATGATCATATTTAT (2688)
TTATTATAT----TTATGTGTATTATTACTGGTCTATCAATTTGTCCTATTATT---GTATTGCCTG-
TCAAGGAAT AAATTGTATGATGATCATATTTAT (2688)
TTATTATAT----TTATGTGTATTATTACTGGTCTATCAATTTGTCCTATTATT---GTATTGCCTG-
TCAAGGAAT AAATTGTATGATGATCATATTTAT (2737)
GCATAGATAGGATCGGATGAGTAGGTTCACTTGCTTGAGTTCACCGGTATAATTCCGG-------AT-
ACATCTGGT DAS-CMS33 TAGGTCATCCTTTGGTCAGCTGCC (2737)
GCATAGATAGGATCGGATGAGTAGGTTCACTTGCTTGAGTTCACCGGTATAATTCCGG-------AT-
ACATCTGGT TAGGTCATCCTTTGGTCAGCTGCC (2726)
GCATAGATAGGA-----TGAGTAGGTTCACTTGCTTGAGTTCACCGGTATAATTCTGG-------AT-
ACATCTGGT TAGGTCATCCTTTGGTCAGCTGCC (2781)
GCATAGATAGGA-----TGAGTAGGTTCACTTGCTTGAGTTCACCGGTATAATTCTGGTTTCTGGAT-
ACATCTGGT TAGGTCAGCCTTTGGTCAGCTGCC (2781)
GCATAGATAGGA-----TGAGTAGGTTCACTTGCTTGAGTTCACCGGTATAATTCTGGTTTCTGGAT-
ACATCTGGT TAGGTCAGCCTTTGGTCAGCTGCC (2781)
GCATAGATAGGA-----TGAGTAGGTTCACTTGCTTGAGTTCACCGGTATAATTCTGGTTTCTGGAT-
ACATCTGGT TAGGTCAGCCTTTGGTCAGCTGCC (2830)
CGCAAGCTTAACTCCGTGCGATATACAATATACAGATTTTATTATGGTTTTCCCCTGAACCTTCGTG-
ACTAACTAT GTTATCATTTTTATAGCTTTATAG (2830)
CGCAAGCTTAACTCCGTGCGATATACAATATACAGATTTTATTATGGTTTTCCCCTGAACCTTCGTG-
ACTAACTAT GTTATCATTTTTATAGCTTTATAG (2814)
CGCAA---------CGTGCGATATACAATATACATATTTTATTATGTTTTT----------TTCGTG-
ACTAACTAT GTTATCATTTTTATAGCTTTATAG (2876)
CGCAAGCTTAACTCCGTGCGATATACACTATACAAATTTTATTATGTTTTT----------TTCGTG-
ACTAACTAT GTTATCATTTTTATAGCTTTATAG (2876)
CGCAAGCTTAACTCCGTGCGATATACACTATACAAATTTTATTATGTTTTT----------TTCGTG-
ACTAACTAT GTTATCATTTTTATAGCTTTATAG (2876)
CGCAAGCTTAACTCCGTGCGATATACACTATACAAATTTTATTATGTTTTT----------TTCGTG-
ACTAACTAT GTTATCATTTTTATAGCTTTATAG DAS-CMS34 (2930)
TCTACAAACTGTTTTATACTCAGCTTGATAAGTACATTCTGGTTTGGACGATGG-TTTTTTTTTCTT-
GCAAAA-TG AATTTGTCTTCAGCCTTTACGACT (2930)
TCTACAAACTGTTTTATACTCAGCTTGATAAGTACATTCTGGTTTGGACGATGG-TTTTTTTTTCTT-
GCAAAA-TG AATTTGTCTTCAGCCTTTACGACT
(2895)
TCTACAAACTGTTTTATACTCAGCTTGATAAGTACATTCTGGTTTGGACGAT----TTTTTTTTCTT-
GCAAAAATG AATTTGTCTTCAGCCTTTACGACT (2966)
TCTACAAACTGTTTTATACTCAGCTTGATAAGTACATTCTGGTTTGGACGATGGTTTTTTTTTTCTT-
GCAAAAATG AATTTGTCTTCAGCCTTTACGACT (2966)
TCTACAAACTGTTTTATACTCAGCTTGATAAGTACATTCTGGTTTGGACGATGGTTTTTTTTTTCTT-
GCAAAAATG AATTTGTCTTCAGCCTTTACGACT (2966)
TCTACAAACTGTTTTATACTCAGCTTGATAAGTACATTCTGGTTTGGACGATGGTTTTTTTTTTCTT-
GCAAAAATG AATTTGTCTTCAGCCTTTACGACT (3028)
ACATACAGTTTAGTT----------------TGTATTAATTGATACCGGAAGATCAGATTCGGACCA-
CATATAAAC AAGGAATATATAGCACGTACTCGC (3028)
ACATACAGTTTAGTT----------------TGTATTAATTGATACCGGAAGATCAGATTCGGACCA-
CATATAAAC AAGGAATATATAGCACGTACTCGC (2991)
ACATACAGTTTAGTTCTTAGAGTATCTCATCTGTATTAATTGATACCGGAAGA---GATTCGGGCCA-
CATATAAAC AAGGAATATATAGCACGTACTCGC (3066)
ACATACAGTTTAGTT----------------TGTATTAATTGATACCAGAAGATCAGATTCGGACCA-
CATATAAAC AAGGAATATATAGCACGTACTCGC (3066)
ACATACAGTTTAGTT----------------TGTATTAATTGATACCAGAAGATCAGATTCGGACCA-
CATATAAAC AAGGAATATATAGCACGTACTCGC (3066)
ACATACAGTTTAGTT----------------TGTATTAATTGATACCAGAAGATCAGATTCGGACCA-
CATATAAAC AAGGAATATATAGCACGTACTCGC (SEQ ID NO: 5) (3112)
TGAACCTTAAATATAGTCAGGAAAATAGAGGGTTAACTAAACCGATCCAGAAACCAATTACATTGAT-
ATTGACTCT ATTCTTCGTT (SEQ ID NO: 6) (3112)
TGAACCTTAAATATAGTCAGGAAAATAGAGGGTTAACTAAACCGATCCAGAAACCAATTACATTGAT-
ATTGACTCT ATTCTTCGTT (SEQ ID NO: 7) (3088)
TGAACCTTAAATATAGTCAGGAACATAGAGGGTTAACTAAACCGATCCAGAAACCAATTACATTGAT-
ATTGACTCT ATTCTTCGTT (SEQ ID NO: 8) (3150)
TGAACCTTAAATATAGTCAGGAACATAGAGGGTTAACTAAACCGATCCAGAAACCAATTACATTGAT-
ATTGACTCT ATTCTTCGTT (SEQ ID NO: 9) (3150)
TGAACCTTAAATATAGTCAGGAACATAGAGGGTTAACTAAACCGATCCAGAAACCAATTACATTGAT-
ATTGACTCT ATTCTTCGTT (SEQ ID NO: 10) (3150)
TGAACCTTAAATATAGTCAGGAACATAGAGGGTTAACTAAACCGATCCAGAAACCAATTACATTGAT-
ATTGACTCT ATTCTTCGTT
The translation start and stop codons and positions for markers
DAS-CMS21 through DAS-CMS35 within the gene are underlined and/or
labeled.
[0062] Gene Specific Assay Design and Validation.
[0063] Alignments of predicted Rf4-bHLH protein sequences indicated
that all three restorer lines had identical protein sequences and
non-restorer lines were identical (data not shown). There were four
amino-acid changes between restorer lines and non-restorer lines:
His (H.sub.103) to Asn (N.sub.103), Ala (A.sub.130) insertion, Pro
(P.sub.266) to Leu (L.sub.267) and Tyr (Y.sub.186) to Phe
(F.sub.187) substitution in the restorer lines. In comparison with
other monocot orthologs, the Phe (F.sub.187) substitution in the
maize restorer allele was conserved, and the other three amino acid
changes were less conserved and were located in variable sites
(data not shown). This conserved substitution was used for gene
specific TaqMan.RTM. assay design.
TABLE-US-00005 SEQ ID NO: 6 primer sequences for an CMS-C line
Forward primer 5'-CAACGGCGTCGAGAAGAAG-3' (SEQ ID NO: 11) VIC
Reporter 5'-CTCGGCGTCGGCCGCGACGAAGAG-3' (SEQ ID NO: 12)
rf4(non-restorer)probe-MGB 5'-ACCGAGAAGTACACCGC-3' (SEQ ID NO: 13)
Reverse primer 5'-ATTGCAACCCATACTCCACGTA-3' (SEQ ID NO: 14) SEQ ID
NO: 8 primer sequences for an Rf4 line Forward primer
5'-CAACGGCGTCGAGAAGAAG-3' (SEQ ID NO: 15) FAM Reporter
5'-TCGGCGTCGGCCGCGACGAAGAG-3' (SEQ ID NO: 16) Rf4 (restorer)
specific 5'-CACCGAGAAGTTTACGGC-3' (SEQ ID NO: 17) probe-MGB Reverse
primer 5'-ATTGCAACCCATACTCCACGTA-3' (SEQ ID NO: 18)
Three known Rf4 restoration lines and six non-restoration lines
were used for assay testing. One hemizygous sample was made by
combining equal amount of DNA from Rf4 line with a CMS-C line.
Real-time PCR was used to test the efficiency of the assay.
Oligonucleotides specific to the Rf4 gene and to the corresponding
CMS-C line or non-restorer were combined in the same assay. FAM was
used to monitor the Rf4 amplicon from the restorer lines and VIC
from non-restorer CMS-C or non-restorer lines. Exponential
amplification phase was observed from cycles 23 to 35 for both Rf4
restorer lines and non-restorer CMS-C or non-restorer lines (FIGS.
1A and 1B).
[0064] Validation of End-Point TaqMan.RTM. Zygosity Analysis.
[0065] An F2 CMS-C/restoration mapping population with 500
individuals was used to validate the assay using end-point
TaqMan.RTM. PCR instead of using real-time TaqMan.RTM. PCR. The
advantages of end-point TaqMan.RTM. over real-time TaqMan.RTM.
include its ease to use and high throughput. For end-point
TaqMan.RTM. PCR, any regular PCR machine that can fit 96- or
384-well plates plus a plate reader that can read FAM and VIC are
sufficient to perform the assay.
[0066] Following completion of the TaqMan.RTM. PCR and fluorescence
reading, the raw fluorescence intensity data directly from the
plate reader were analyzed in the KLIMS system. A graph with RFU
(relative fluorescence unit) of FAM as x-axis and VIC as y-axis was
generated. Zygosity calls were made based on the cluster separation
in a cluster view (FIG. 2). Since FAM was used to monitor the
amplification of Rf4 from restorer lines and VIC for
non-restorer/CMS-C lines, samples with strong signals of FAM and
little or no VIC are homozygous for Rf4 allele; samples with strong
signals of VIC and little or no FAM are non-restorer/CMS-C lines
(nulls); and samples with strong signals of both FAM and VIC are
hemizygous for Rf4 allele.
[0067] F2 genotypic data based on the Rf4 gene specific end-point
TaqMan.RTM. PCR assay matched completely with field phenotypic
data, which demonstrated the effectiveness and accuracy of the
end-point TaqMan.RTM. PCR assay for testing Rf4 zygosity in a high
throughput way. This assay enables large scale and high throughput
screening of maize germplasms with the Rf4 restoration gene. This
assay will also increase the scale of using a CMS-C/Rf4 system for
hybrid corn seed production.
Sequence CWU 1
1
18119DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1caacggcgtc gagaagaag 19222DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2taacgttggg tatgaggtgc at 22317DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 3accgagaagt acaccgc
17418DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 4caccgagaag tttacggc 1853197DNAZea mays 5ggcaagctaa
tggggtacat atggaaggag gaaaccaagt cgatcgtcgt cgtagcatgt 60cggtgtgggt
actacactac acacacatat acatgggcaa cgcaaggcca cctttctgaa
120tcctgcatga gcgtgtacca ctagaattgt cagtgtgtgc ggtgtatggc
aggtttttgg 180ttcggcaagt ggggccctcc ggggaggaat ctcagtaaca
aaccgctctt ctgaaaaggt 240cagccatccc cggtccggtc cggtgatgtc
gtcgctgtcg ctctgctagc ttgctgccga 300tccccccccc cccccccccc
cttcttctct ctacccctcc ctccacctca taaatactta 360gtttaataac
cttgcactgc cgcagtagcc cttaactgct gctatctatc tcttttctga
420aggaaaaaaa aggtttgata ctcctctacc tagctagtcc tgcatgccgc
taatgtgcgt 480cttgcctgtt tatttgttct taataagggc tgcctatcta
ttatattttg cacctgtttt 540gctgtgttct tggtaactag cttaattcct
tcgcctacaa tcgtcaaatc ccccccatca 600tcagtcagat gaacttttga
tcgaattgaa gttgttcttc taattcggcc ccagcagcgc 660ccatgcatct
ggttttattt gctttctgtt gggtataata tgcaagacct tttgttgcta
720gggcaaggct gcaaccacat gcgtgtactg aactcatgat gtaactcatc
ctttttgttt 780gctcacagaa tcactactct actgcacttc cttttcatcc
gatccgcaat cttttttttc 840ttttacatgc tttagttttc tctctttctt
gattacaaac atgattactg gaactttctt 900aggctgcctt ccccttcctt
ggatctgctt tagttttctt ttttgggcta ccgcgcgcgg 960cttatttgag
tttatcactt gctgcatata cataatatat atatacatgc atgcgatggc
1020gttcatgtta ctcaactaca gatctgtttc tgttcgtgtg tttcagttca
gcgcgcagtt 1080aagcatagca ggacgaccac gacgatgtat cacccgcagt
gcgagctcct gacgatggcg 1140cacgaaacgc cggacctgga cgccggccag
ccgcacctaa ccgtctccgg cgtcgccagc 1200atcccggcag agctgagctt
ccacctgctg cactcgctcg acgccgcggc ggcggtcaat 1260cccgtcacgg
cgccgccgca gtccaccatc gactacttcc tcggcggcgc cgatccccac
1320cagcaggcca tgcagtacga gccgctgccg cccgccgcgg gcggccacca
ccagtacacc 1380atggacatgt tccgcgacta ctgcgacggc cactacccca
ccgccgagcc gtacatccgc 1440gggacaatga ctggagccct cgtgttcggg
gccaccgacg acgacgactc ggccgctgcc 1500tacatgcccg gggggcactt
tgagacctcc ccgccgccgc cacgcgccac cggccgcggc 1560aggaagcggg
gcagggcgct gggcggcggc ttccatgctg tgctggccaa cggcgtcgag
1620aagaaggaga agcagcgccg gctgcggctc accgagaagt acaccgccct
catgcacctc 1680atacccaacg ttacaaaggt cgtaccaaat cctcctctta
tgttcgtcca tcgtttcaaa 1740ttaagttaaa aaattaattc acggttcttg
ttgtttattt tttgcgcact gcagactgat 1800agggcgacgg tgatctcgga
cgcgatcgag tacatccagg agctggggag gacggtggag 1860gagctgacgc
tgctggtgga gaagaagcgg cgccggaggg agctgcaggg ggacgtcgtg
1920gacgcggcgc cggctgcggt ggttgctgcc gccggtgagg cggagagctc
ggagggcgag 1980gtggctcctc cgccgccggc cgtgccgcgg cagccgatcc
ggagcacgta catccagcgg 2040cggagcaagg acacgtccgt ggacgtgcgg
atcgtggagg aggacgtgaa catcaagctc 2100accaagcgcc ggcgcgacgg
gtgcctcgca gccgcgtcgc gcgcgctgga tgacctccgc 2160cttgacctcg
tccacctctc cggcggcaag atcggtgact gtcaaatcta catgttcaac
2220accaaggtac atacgaatac gatacgtagc cattgatcga tctgtaattc
tgtagcctga 2280cgattccgag gtttctggtg ctaaaaaatg catctttttt
tctcagatga caatgctttc 2340tgtctttgtt caccgcagat tcacaagggg
tcttcagtgt ttgcgagtgc agtggccggt 2400aggctgatgg aagtggtgga
cgagtactag gctaccatgc acttgaattt ctagctagct 2460ctacgtaccg
cgctgctatg aatctagcta tagcgtttct tggatgaaag actagttagt
2520tgttaccttc tatctttgct tcaattaaat ccgcttgctc gttacagact
gagtttgttt 2580ctaaatgtca aggttgtttt ggtcaaattg aataaattgg
cacactggcc tgtgaggtta 2640ttatatatat ttatgtgttt attactggtc
tattaatttg tcttattatt aatgtattgc 2700ctgtcaagga ataaatggta
tgatgaccat atttatgcat agataggatc ggatgagtag 2760gttcacttgc
ttgagttcac cggtataatt ccggatacat ctggttaggt catcctttgg
2820tcagctgccc gcaagcttaa ctccgtgcga tatacaatat acagatttta
ttatggtttt 2880cccctgaacc ttcgtgacta actatgttat catttttata
gctttatagt ctacaaactg 2940ttttatactc agcttgataa gtacattctg
gtttggacga tggttttttt ttcttgcaaa 3000atgaatttgt cttcagcctt
tacgactaca tacagtttag tttgtattaa ttgataccgg 3060aagatcagat
tcggaccaca tataaacaag gaatatatag cacgtactcg ctgaacctta
3120aatatagtca ggaaaataga gggttaacta aaccgatcca gaaaccaatt
acattgatat 3180tgactctatt cttcgtt 319763197DNAZea mays 6ggcaagctaa
tggggtacat atggaaggag gaaaccaagt cgatcgtcgt cgtagcatgt 60cggtgtgggt
actacactac acacacatat acatgggcaa cgcaaggcca cctttctgaa
120tcctgcatga gcgtgtacca ctagaattgt cagtgtgtgc ggtgtatggc
aggtttttgg 180ttcggcaagt ggggccctcc ggggaggaat ctcagtaaca
aaccgctctt ctgaaaaggt 240cagccatccc cggtccggtc cggtgatgtc
gtcgctgtcg ctctgctagc ttgctgccga 300tccccccccc cccccccccc
cttcttctct ctacccctcc ctccacctca taaatactta 360gtttaataac
cttgcactgc cgcagtagcc cttaactgct gctatctatc tcttttctga
420aggaaaaaaa aggtttgata ctcctctacc tagctagtcc tgcatgccgc
taatgtgcgt 480cttgcctgtt tatttgttct taataagggc tgcctatcta
ttatattttg cacctgtttt 540gctgtgttct tggtaactag cttaattcct
tcgcctacaa tcgtcaaatc ccccccatca 600tcagtcagat gaacttttga
tcgaattgaa gttgttcttc taattcggcc ccagcagcgc 660ccatgcatct
ggttttattt gctttctgtt gggtataata tgcaagacct tttgttgcta
720gggcaaggct gcaaccacat gcgtgtactg aactcatgat gtaactcatc
ctttttgttt 780gctcacagaa tcactactct actgcacttc cttttcatcc
gatccgcaat cttttttttc 840ttttacatgc tttagttttc tctctttctt
gattacaaac atgattactg gaactttctt 900aggctgcctt ccccttcctt
ggatctgctt tagttttctt ttttgggcta ccgcgcgcgg 960cttatttgag
tttatcactt gctgcatata cataatatat atatacatgc atgcgatggc
1020gttcatgtta ctcaactaca gatctgtttc tgttcgtgtg tttcagttca
gcgcgcagtt 1080aagcatagca ggacgaccac gacgatgtat cacccgcagt
gcgagctcct gacgatggcg 1140cacgaaacgc cggacctgga cgccggccag
ccgcacctaa ccgtctccgg cgtcgccagc 1200atcccggcag agctgagctt
ccacctgctg cactcgctcg acgccgcggc ggcggtcaat 1260cccgtcacgg
cgccgccgca gtccaccatc gactacttcc tcggcggcgc cgatccccac
1320cagcaggcca tgcagtacga gccgctgccg cccgccgcgg gcggccacca
ccagtacacc 1380atggacatgt tccgcgacta ctgcgacggc cactacccca
ccgccgagcc gtacatccgc 1440gggacaatga ctggagccct cgtgttcggg
gccaccgacg acgacgactc ggccgctgcc 1500tacatgcccg gggggcactt
tgagacctcc ccgccgccgc cacgcgccac cggccgcggc 1560aggaagcggg
gcagggcgct gggcggcggc ttccatgctg tgctggccaa cggcgtcgag
1620aagaaggaga agcagcgccg gctgcggctc accgagaagt acaccgccct
catgcacctc 1680atacccaacg ttacaaaggt cgtaccaaat cctcctctta
tgttcgtcca tcgtttcaaa 1740ttaagttaaa aaattaattc acggttcttg
ttgtttattt tttgcgcact gcagactgat 1800agggcgacgg tgatctcgga
cgcgatcgag tacatccagg agctggggag gacggtggag 1860gagctgacgc
tgctggtgga gaagaagcgg cgccggaggg agctgcaggg ggacgtcgtg
1920gacgcggcgc cggctgcggt ggttgctgcc gccggtgagg cggagagctc
ggagggcgag 1980gtggctcctc cgccgccggc cgtgccgcgg cagccgatcc
ggagcacgta catccagcgg 2040cggagcaagg acacgtccgt ggacgtgcgg
atcgtggagg aggacgtgaa catcaagctc 2100accaagcgcc ggcgcgacgg
gtgcctcgca gccgcgtcgc gcgcgctgga tgacctccgc 2160cttgacctcg
tccacctctc cggcggcaag atcggtgact gtcaaatcta catgttcaac
2220accaaggtac atacgaatac gatacgtagc cattgatcga tctgtaattc
tgtagcctga 2280cgattccgag gtttctggtg ctaaaaaatg catctttttt
tctcagatga caatgctttc 2340tgtctttgtt caccgcagat tcacaagggg
tcttcagtgt ttgcgagtgc agtggccggt 2400aggctgatgg aagtggtgga
cgagtactag gctaccatgc acttgaattt ctagctagct 2460ctacgtaccg
cgctgctatg aatctagcta tagcgtttct tggatgaaag actagttagt
2520tgttaccttc tatctttgct tcaattaaat ccgcttgctc gttacagact
gagtttgttt 2580ctaaatgtca aggttgtttt ggtcaaattg aataaattgg
cacactggcc tgtgaggtta 2640ttatatatat ttatgtgttt attactggtc
tattaatttg tcttattatt aatgtattgc 2700ctgtcaagga ataaatggta
tgatgaccat atttatgcat agataggatc ggatgagtag 2760gttcacttgc
ttgagttcac cggtataatt ccggatacat ctggttaggt catcctttgg
2820tcagctgccc gcaagcttaa ctccgtgcga tatacaatat acagatttta
ttatggtttt 2880cccctgaacc ttcgtgacta actatgttat catttttata
gctttatagt ctacaaactg 2940ttttatactc agcttgataa gtacattctg
gtttggacga tggttttttt ttcttgcaaa 3000atgaatttgt cttcagcctt
tacgactaca tacagtttag tttgtattaa ttgataccgg 3060aagatcagat
tcggaccaca tataaacaag gaatatatag cacgtactcg ctgaacctta
3120aatatagtca ggaaaataga gggttaacta aaccgatcca gaaaccaatt
acattgatat 3180tgactctatt cttcgtt 319773173DNAZea mays 7ggcaagctaa
tggggtacat atggaaggag gaaaccaagt cgatcgtcgt cgtagcatgt 60cggtgtgggt
actacactac acacacatat acatgggcaa cgcaaggcca cctttctgaa
120tcctgcatga gcgtgtacca ctagaattgt cagtgtgtgc ggtgtatggc
aggtttttgg 180ttcggcaagt ggggccctcc ggggaggaat ctcagtaaca
aaccgctctt ctgaaaaggt 240cagccatccc cggtccggtc cggtgatgtc
gtcgctgtcg ctctgctagc ttgctgccga 300tccccccccc ttcttctctc
tacccctccc tccacctcat aaatacttag tttaataacc 360ttgcactgcc
gcagtagccc ttaactgctg ctatctatct cttttctgaa ggaaaaaaaa
420ggtttgatac tcctctacct agctagtcct gcatgccgct aatgtgcgtc
ttgcctgttt 480atttgttctt aataagggct gcctatctat tatattttgc
acctgttttg ctgtgttctt 540ggtaactagc ttaattcctt tgcctacaat
cgtcaaatcc cccccatcat cagtcagatg 600aacttttgat cgaattgaag
ttgttcttct aattcggccc cagcagcgcc catgcatctg 660gttttatttg
ctttctgttg ggtataatat gcaagacctt ttgttgctag ggcaaggctg
720caaccacatg cgtgtactga actcatgatg taactcatcc tttttgtttg
ctcacagaat 780cactactcta ctgcacttcc ttttcatccg atccgcaatc
ttttttttct tttacatgct 840ttagttttct ctctttcttg attacaaaca
tgattactgg aactttctta ggctgccttc 900cccttccttg gatctgcttt
agttttcttt tttgggctac cgcgcgcggc ttatttgagt 960ttatcacttg
ctgcatatac ataatatata tatacatgca tgcgatggcg ttcatgttac
1020tcaactacag atctgtttct gttcgtgtgt ttcagttcag cgcgcagtta
agcatagcag 1080gacgaccacg acgatgtatc acccgcagtg cgagctcctg
acgatggcgc acgaaacgcc 1140ggacctggac gccggccagc cgcacctaac
cgtctccggc gtcgccagca tcccggcaga 1200gctgagcttc cacctgctgc
actcgctcga cgccgcggcg gcggtcaatc ccgtcacggc 1260gccgccgcag
tccaccatcg actacttcct cggcggcgcc gatccccacc agcaggccat
1320gcagtacgag ccgctgccgc ccgccgcggg cggccaccac cagtacacca
tggacatgtt 1380ccgcgactac tgcgacggcc actaccccac cgccgagccg
tacatccgcg ggacaatgac 1440tggagccctc gtgttcgggg ccaccgacga
cgacgactcg gccgctgcct acatgcccgg 1500ggggcacttt gagacctccc
cgccgccgcc acgcgccacc ggccgcggca ggaagcgggg 1560cagggcgctg
ggcggcggct tccatgctgt gctggccaac ggcgtcgaga agaaggagaa
1620gcagcgccgg ctgcggctca ccgagaagta caccgccctc atgcacctca
tacccaacgt 1680tacaaaggtc gtaccaaatc ctcctcttat gttcgtccat
cgtttgaaat taagttaaaa 1740aattaattca cggttcttgt tgtttatttt
ttgcgcactg cagactgata gggcgacggt 1800gatctcggac gcgatcgagt
acatccagga gctggggagg acggtggagg agctgacgct 1860gctggtggag
aagaagcggc gccggaggga gctgcagggg gacgtcgtgg acgcggcgcc
1920ggctgcggtg gttgctgccg ccggtgaggc ggagagctcg gagggcgagg
tggctcctcc 1980gccgccggcc gtgccgcggc agccgatccg gagcacgtac
atccagcggc ggagcaagga 2040cacgtccgtg gacgtgcgga tcgtggagga
ggacgtgaac atcaagctca ccaagcgccg 2100gcgcgacggg tgcctcgcag
ccgcgtcgcg cgcgctggat gacctccgcc ttgacctcgt 2160ccacctctcc
ggcggcaaga tcggtgactg tcaaatctac atgttcaaca ccaaggtaca
2220tacgaatacg atacgtagcc attgatcgat ctgtaattct gtagcctgac
gattccgagg 2280tttctggtgc taaaaaatgc atcttttttt ctcagatgac
aatgctttct gtctttgttc 2340accgcagatt cacaaggggt cttcagtgtt
tgcgagtgca gtggccggta ggctgatgga 2400agtggtggac gagtactagg
ctaccatgca cttgaatttc tagctagctc tacgtaccgc 2460gctgctatga
atctagctat agcgtttctt ggatgaaaga ctagttagtt gttaccttct
2520atctttgctt caattaaatc cgcttgctcg ttacagactg agtttgtttc
taaatgtcaa 2580ggttgttttg gtcaaattga ataaattggc acactggcct
gtgaggttat tatatatatt 2640tatgtgttta ttactggtct attaatttgt
cctattatta atgtattgcc tgtcaaggaa 2700taaatgatat gatgaccata
tttatgcata gataggatga gtaggttcac ttgcttgagt 2760tcaccggtat
aattctggat acatctggtt aggtcatcct ttggtcagct gcccgcaacg
2820tgcgatatac aatatacata ttttattatg tttttttcgt gactaactat
gttatcattt 2880ttatagcttt atagtctaca aactgtttta tactcagctt
gataagtaca ttctggtttg 2940gacgattttt ttttcttgca aaaatgaatt
tgtcttcagc ctttacgact acatacagtt 3000tagttcttag agtatctcat
ctgtattaat tgataccgga agagattcgg gccacatata 3060aacaaggaat
atatagcacg tactcgctga accttaaata tagtcaggaa catagagggt
3120taactaaacc gatccagaaa ccaattacat tgatattgac tctattcttc gtt
317383235DNAZea mays 8ggcaagctaa tggggtacat atggaaggag gaaaccaagt
cgatcgtcgt cgtagcatgt 60cggtgtgggt actacactac acacacatat acatgggcaa
cgcaaggcca cctttctgaa 120tcctgcatga gcgtgtacca ctagaattgt
cagtgtgtgc ggtgtatggc aggtttttgg 180ttcggcaagt ggggccctcc
ggggaggaat ctcagtaaca aaccgctctt ctgaaaaggt 240cagccatccc
cggtccggtc cggtgatgtc gtcgctgtcg ctctgctagc ttgctgccga
300tccccccccc cccccccccc cttcttctct ctacccctcc ctccacctca
taaatactta 360gtttaataac cttgcactgc cgcagtagcc cttaactgct
gctatctatc tcttttctga 420aggaaaaaaa aggtttgata ctcctctacc
tagctagtcc tgcatgccgc taatgtgcgt 480cttgcctgtt tatttgttct
taataagggc tgcctatcta ttatattttg cacctgtttt 540gctgtgttct
tggtaactag cttaattcct ttgcctacaa tcgtcaaatc ccccccatca
600tcagtcagat gaacttttga tcgaattgaa gttgttcttc taattcggcc
ccagcagcgc 660ccatgcatct ggttttattt gctttctgtt gggtataata
tgcaagacct tttgttgcta 720gggcaaggct gcaaccacat gcgtgtactg
aactcatgat gtaactcatc ctttttgttt 780gctcacagaa tcactactct
actgcacttc cttttcatcc gatccgcaat cttttttttc 840ttttacatgc
tttagttttc tctctttctt gattacaaac atgattactg gaactttctt
900aggctgcctt ccccttcctt ggatctgctt tagttttctt ttttgggcta
ccgcgcgcgg 960cttatttgag tttatcactt gctgcatata cataatatat
atatacatgc atgcgatggc 1020gttcatgtta ctcaactaca gatctgtttc
tgttcgtgtg tttcagttca gcgcgcagtt 1080aagcatagca ggacgaccac
gacgatgtat cacccgcagt gcgagctcct gacgatggcg 1140cacgaaacgc
cggacctgga cgccggccag ccgcacctaa ccgtctccgg cgtcgccagc
1200atcccggcag agctgagctt ccacctgctg cactcgctcg acgccgcggc
ggcggtcaat 1260cccgtcacgg cgccgccgca gtccaccatc gactacttcc
tcggcggcgc cgatccccac 1320cagcaggcca tgcagtacga gccgctgccg
cccgccgcgg gcggccacca ccagtacacc 1380atggacatgt tccgcgacta
ctgcgacggc aactacccca ccgccgagcc gtacatccgc 1440gggacaatga
ctggagccct cgtgttcggg gccaccgacg acgacgactc ggccgctgcc
1500gcctacatgc ccggggggca ctttgagacc tccccgccgc cgccacgcgc
caccggccgc 1560ggcaggaagc ggggcagggc gctgggcggc ggcttccatg
ctgtgctggc caacggcgtc 1620gagaagaagg agaagcagcg ccggctgcgg
ctcaccgaga agtttacggc cctcatgcac 1680ctcataccca acgttacgaa
ggtcgtacgg cgtacttgcg cgcggaccaa atcctcctct 1740tatgttcgtc
gtccatcgtc tcaaattaat tcacggttct tgttgttgtt tattttttgc
1800gcactgcaga ctgatagggc gacggtgatc tcggacgcga tcgagtacat
ccaggagctg 1860gggaggacgg tggaggagct gacgctgctg gtggagaaga
agcggcgccg gagggagctg 1920cagggggacg tcgtggacgc ggcgccggct
gcggtggttg ctgccgccgg tgaggcggag 1980agctcggagg gcgaggtggc
tcctccgccg ctggccgtgc cgcggcagcc gatccggagc 2040acgtacatcc
agcggcggag caaggacacg tccgtggacg tgcggatcgt ggaggaggac
2100gtgaacatca agctcaccaa gcgccggcgc gacgggtgcc tcgcagccgc
gtcgcgcgcg 2160ctggacgacc tccgccttga cctcgtccac ctctccggcg
gcaagatcgg tgactgtcaa 2220atctacatgt tcaacaccaa ggtacatacg
aatacgatac gtagccattg atcgatctgt 2280aattctgtag cctgacgatt
tcatgcatta cttttccgag gtttctgtgc tatactacct 2340aacctaggtg
ctaaaaaatg cacctttttt tctcagatga caatgctttc tgtctttgtt
2400caccgcagat tcacaagggg tcttcagtgt ttgcgagtgc agtggccggt
aggctgatgg 2460aagtggtgga cgagtactag gctaccatgc acttgaattt
ctagctagct ctacgtaccg 2520cgctgctatg aatctagcta tagcgtttct
tggatgaaag aatagttagt tgttaccttc 2580tatctttgct tcaattaaat
ccgcttgctc gttacagact gagtttgttt ctaaatgtca 2640aggttgtttt
ggtcaaattg aataaattgg cacactggcc tgtgaggtta ttatatttat
2700gtgtattatt actggtctat caatttgtcc tattattgta ttgcctgtca
aggaataaat 2760tgtatgatga tcatatttat gcatagatag gatgagtagg
ttcacttgct tgagttcacc 2820ggtataattc tggtttctgg atacatctgg
ttaggtcagc ctttggtcag ctgcccgcaa 2880gcttaactcc gtgcgatata
cactatacaa attttattat gtttttttcg tgactaacta 2940tgttatcatt
tttatagctt tatagtctac aaactgtttt atactcagct tgataagtac
3000attctggttt ggacgatggt tttttttttc ttgcaaaaat gaatttgtct
tcagccttta 3060cgactacata cagtttagtt tgtattaatt gataccagaa
gatcagattc ggaccacata 3120taaacaagga atatatagca cgtactcgct
gaaccttaaa tatagtcagg aacatagagg 3180gttaactaaa ccgatccaga
aaccaattac attgatattg actctattct tcgtt 323593235DNAZea mays
9ggcaagctaa tggggtacat atggaaggag gaaaccaagt cgatcgtcgt cgtagcatgt
60cggtgtgggt actacactac acacacatat acatgggcaa cgcaaggcca cctttctgaa
120tcctgcatga gcgtgtacca ctagaattgt cagtgtgtgc ggtgtatggc
aggtttttgg 180ttcggcaagt ggggccctcc ggggaggaat ctcagtaaca
aaccgctctt ctgaaaaggt 240cagccatccc cggtccggtc cggtgatgtc
gtcgctgtcg ctctgctagc ttgctgccga 300tccccccccc cccccccccc
cttcttctct ctacccctcc ctccacctca taaatactta 360gtttaataac
cttgcactgc cgcagtagcc cttaactgct gctatctatc tcttttctga
420aggaaaaaaa aggtttgata ctcctctacc tagctagtcc tgcatgccgc
taatgtgcgt 480cttgcctgtt tatttgttct taataagggc tgcctatcta
ttatattttg cacctgtttt 540gctgtgttct tggtaactag cttaattcct
ttgcctacaa tcgtcaaatc ccccccatca 600tcagtcagat gaacttttga
tcgaattgaa gttgttcttc taattcggcc ccagcagcgc 660ccatgcatct
ggttttattt gctttctgtt gggtataata tgcaagacct tttgttgcta
720gggcaaggct gcaaccacat gcgtgtactg aactcatgat gtaactcatc
ctttttgttt 780gctcacagaa tcactactct actgcacttc cttttcatcc
gatccgcaat cttttttttc 840ttttacatgc tttagttttc tctctttctt
gattacaaac atgattactg gaactttctt 900aggctgcctt ccccttcctt
ggatctgctt tagttttctt ttttgggcta ccgcgcgcgg 960cttatttgag
tttatcactt gctgcatata cataatatat atatacatgc atgcgatggc
1020gttcatgtta ctcaactaca gatctgtttc tgttcgtgtg tttcagttca
gcgcgcagtt 1080aagcatagca ggacgaccac gacgatgtat cacccgcagt
gcgagctcct gacgatggcg 1140cacgaaacgc cggacctgga cgccggccag
ccgcacctaa ccgtctccgg cgtcgccagc 1200atcccggcag agctgagctt
ccacctgctg cactcgctcg acgccgcggc ggcggtcaat 1260cccgtcacgg
cgccgccgca gtccaccatc gactacttcc tcggcggcgc cgatccccac
1320cagcaggcca tgcagtacga gccgctgccg cccgccgcgg gcggccacca
ccagtacacc 1380atggacatgt tccgcgacta ctgcgacggc aactacccca
ccgccgagcc gtacatccgc 1440gggacaatga ctggagccct cgtgttcggg
gccaccgacg acgacgactc ggccgctgcc 1500gcctacatgc ccggggggca
ctttgagacc tccccgccgc cgccacgcgc caccggccgc
1560ggcaggaagc ggggcagggc gctgggcggc ggcttccatg ctgtgctggc
caacggcgtc 1620gagaagaagg agaagcagcg ccggctgcgg ctcaccgaga
agtttacggc cctcatgcac 1680ctcataccca acgttacgaa ggtcgtacgg
cgtacttgcg cgcggaccaa atcctcctct 1740tatgttcgtc gtccatcgtc
tcaaattaat tcacggttct tgttgttgtt tattttttgc 1800gcactgcaga
ctgatagggc gacggtgatc tcggacgcga tcgagtacat ccaggagctg
1860gggaggacgg tggaggagct gacgctgctg gtggagaaga agcggcgccg
gagggagctg 1920cagggggacg tcgtggacgc ggcgccggct gcggtggttg
ctgccgccgg tgaggcggag 1980agctcggagg gcgaggtggc tcctccgccg
ctggccgtgc cgcggcagcc gatccggagc 2040acgtacatcc agcggcggag
caaggacacg tccgtggacg tgcggatcgt ggaggaggac 2100gtgaacatca
agctcaccaa gcgccggcgc gacgggtgcc tcgcagccgc gtcgcgcgcg
2160ctggacgacc tccgccttga cctcgtccac ctctccggcg gcaagatcgg
tgactgtcaa 2220atctacatgt tcaacaccaa ggtacatacg aatacgatac
gtagccattg atcgatctgt 2280aattctgtag cctgacgatt tcatgcatta
cttttccgag gtttctgtgc tatactacct 2340aacctaggtg ctaaaaaatg
cacctttttt tctcagatga caatgctttc tgtctttgtt 2400caccgcagat
tcacaagggg tcttcagtgt ttgcgagtgc agtggccggt aggctgatgg
2460aagtggtgga cgagtactag gctaccatgc acttgaattt ctagctagct
ctacgtaccg 2520cgctgctatg aatctagcta tagcgtttct tggatgaaag
aatagttagt tgttaccttc 2580tatctttgct tcaattaaat ccgcttgctc
gttacagact gagtttgttt ctaaatgtca 2640aggttgtttt ggtcaaattg
aataaattgg cacactggcc tgtgaggtta ttatatttat 2700gtgtattatt
actggtctat caatttgtcc tattattgta ttgcctgtca aggaataaat
2760tgtatgatga tcatatttat gcatagatag gatgagtagg ttcacttgct
tgagttcacc 2820ggtataattc tggtttctgg atacatctgg ttaggtcagc
ctttggtcag ctgcccgcaa 2880gcttaactcc gtgcgatata cactatacaa
attttattat gtttttttcg tgactaacta 2940tgttatcatt tttatagctt
tatagtctac aaactgtttt atactcagct tgataagtac 3000attctggttt
ggacgatggt tttttttttc ttgcaaaaat gaatttgtct tcagccttta
3060cgactacata cagtttagtt tgtattaatt gataccagaa gatcagattc
ggaccacata 3120taaacaagga atatatagca cgtactcgct gaaccttaaa
tatagtcagg aacatagagg 3180gttaactaaa ccgatccaga aaccaattac
attgatattg actctattct tcgtt 3235103235DNAZea mays 10ggcaagctaa
tggggtacat atggaaggag gaaaccaagt cgatcgtcgt cgtagcatgt 60cggtgtgggt
actacactac acacacatat acatgggcaa cgcaaggcca cctttctgaa
120tcctgcatga gcgtgtacca ctagaattgt cagtgtgtgc ggtgtatggc
aggtttttgg 180ttcggcaagt ggggccctcc ggggaggaat ctcagtaaca
aaccgctctt ctgaaaaggt 240cagccatccc cggtccggtc cggtgatgtc
gtcgctgtcg ctctgctagc ttgctgccga 300tccccccccc cccccccccc
cttcttctct ctacccctcc ctccacctca taaatactta 360gtttaataac
cttgcactgc cgcagtagcc cttaactgct gctatctatc tcttttctga
420aggaaaaaaa aggtttgata ctcctctacc tagctagtcc tgcatgccgc
taatgtgcgt 480cttgcctgtt tatttgttct taataagggc tgcctatcta
ttatattttg cacctgtttt 540gctgtgttct tggtaactag cttaattcct
ttgcctacaa tcgtcaaatc ccccccatca 600tcagtcagat gaacttttga
tcgaattgaa gttgttcttc taattcggcc ccagcagcgc 660ccatgcatct
ggttttattt gctttctgtt gggtataata tgcaagacct tttgttgcta
720gggcaaggct gcaaccacat gcgtgtactg aactcatgat gtaactcatc
ctttttgttt 780gctcacagaa tcactactct actgcacttc cttttcatcc
gatccgcaat cttttttttc 840ttttacatgc tttagttttc tctctttctt
gattacaaac atgattactg gaactttctt 900aggctgcctt ccccttcctt
ggatctgctt tagttttctt ttttgggcta ccgcgcgcgg 960cttatttgag
tttatcactt gctgcatata cataatatat atatacatgc atgcgatggc
1020gttcatgtta ctcaactaca gatctgtttc tgttcgtgtg tttcagttca
gcgcgcagtt 1080aagcatagca ggacgaccac gacgatgtat cacccgcagt
gcgagctcct gacgatggcg 1140cacgaaacgc cggacctgga cgccggccag
ccgcacctaa ccgtctccgg cgtcgccagc 1200atcccggcag agctgagctt
ccacctgctg cactcgctcg acgccgcggc ggcggtcaat 1260cccgtcacgg
cgccgccgca gtccaccatc gactacttcc tcggcggcgc cgatccccac
1320cagcaggcca tgcagtacga gccgctgccg cccgccgcgg gcggccacca
ccagtacacc 1380atggacatgt tccgcgacta ctgcgacggc aactacccca
ccgccgagcc gtacatccgc 1440gggacaatga ctggagccct cgtgttcggg
gccaccgacg acgacgactc ggccgctgcc 1500gcctacatgc ccggggggca
ctttgagacc tccccgccgc cgccacgcgc caccggccgc 1560ggcaggaagc
ggggcagggc gctgggcggc ggcttccatg ctgtgctggc caacggcgtc
1620gagaagaagg agaagcagcg ccggctgcgg ctcaccgaga agtttacggc
cctcatgcac 1680ctcataccca acgttacgaa ggtcgtacgg cgtacttgcg
cgcggaccaa atcctcctct 1740tatgttcgtc gtccatcgtc tcaaattaat
tcacggttct tgttgttgtt tattttttgc 1800gcactgcaga ctgatagggc
gacggtgatc tcggacgcga tcgagtacat ccaggagctg 1860gggaggacgg
tggaggagct gacgctgctg gtggagaaga agcggcgccg gagggagctg
1920cagggggacg tcgtggacgc ggcgccggct gcggtggttg ctgccgccgg
tgaggcggag 1980agctcggagg gcgaggtggc tcctccgccg ctggccgtgc
cgcggcagcc gatccggagc 2040acgtacatcc agcggcggag caaggacacg
tccgtggacg tgcggatcgt ggaggaggac 2100gtgaacatca agctcaccaa
gcgccggcgc gacgggtgcc tcgcagccgc gtcgcgcgcg 2160ctggacgacc
tccgccttga cctcgtccac ctctccggcg gcaagatcgg tgactgtcaa
2220atctacatgt tcaacaccaa ggtacatacg aatacgatac gtagccattg
atcgatctgt 2280aattctgtag cctgacgatt tcatgcatta cttttccgag
gtttctgtgc tatactacct 2340aacctaggtg ctaaaaaatg cacctttttt
tctcagatga caatgctttc tgtctttgtt 2400caccgcagat tcacaagggg
tcttcagtgt ttgcgagtgc agtggccggt aggctgatgg 2460aagtggtgga
cgagtactag gctaccatgc acttgaattt ctagctagct ctacgtaccg
2520cgctgctatg aatctagcta tagcgtttct tggatgaaag aatagttagt
tgttaccttc 2580tatctttgct tcaattaaat ccgcttgctc gttacagact
gagtttgttt ctaaatgtca 2640aggttgtttt ggtcaaattg aataaattgg
cacactggcc tgtgaggtta ttatatttat 2700gtgtattatt actggtctat
caatttgtcc tattattgta ttgcctgtca aggaataaat 2760tgtatgatga
tcatatttat gcatagatag gatgagtagg ttcacttgct tgagttcacc
2820ggtataattc tggtttctgg atacatctgg ttaggtcagc ctttggtcag
ctgcccgcaa 2880gcttaactcc gtgcgatata cactatacaa attttattat
gtttttttcg tgactaacta 2940tgttatcatt tttatagctt tatagtctac
aaactgtttt atactcagct tgataagtac 3000attctggttt ggacgatggt
tttttttttc ttgcaaaaat gaatttgtct tcagccttta 3060cgactacata
cagtttagtt tgtattaatt gataccagaa gatcagattc ggaccacata
3120taaacaagga atatatagca cgtactcgct gaaccttaaa tatagtcagg
aacatagagg 3180gttaactaaa ccgatccaga aaccaattac attgatattg
actctattct tcgtt 32351119DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 11caacggcgtc gagaagaag
191224DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 12ctcggcgtcg gccgcgacga agag
241317DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 13accgagaagt acaccgc 171422DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
14attgcaaccc atactccacg ta 221519DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 15caacggcgtc gagaagaag
191623DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 16tcggcgtcgg ccgcgacgaa gag
231718DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 17caccgagaag tttacggc 181822DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
18attgcaaccc atactccacg ta 22
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